Tumor targeting probes for image-guided surgical procedures

Abstract

A unique near-infrared (NIR) fluorescent imaging probe, ICG-p28, composed of the clinically non-toxic tumor-targeting peptide p28 and the FDA-approved NIR dye indocyanine green (ICG), has been analyzed for in vivo kinetics and optimized for clinical practice. Intraoperative imaging with ICG-p28 was used to identify small (≤1 mm) lymph node (LN)-containing cancer cells. Image-guided surgery with ICG-p28 reduced residual normalized tumor DNA at the margin site by 6.6x10 compared to a control approach. 3 It demonstrated a greater than fold reduction and resulted in improved tumor recurrence rates (92% specificity) in multiple animal models.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 264,732, filed December 1, 2021, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under Federal Grant Number R01EB023924 awarded by the NIH. The Government has certain rights in the invention.

[0003] Sequence Listing Reference This application contains a Sequence Listing that has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 29, 2022, is named txt_46466-57.txt and is 2229 bytes in size. [Background technology]

[0004] Breast cancer is primarily treated by surgical excision of the tumor with surrounding normal breast tissue margins. In early stages, these procedures take the form of breast-conserving surgery (BCS) or lumpectomy. Although BCS offers satisfactory cosmetic outcomes, the re-excision rate is approximately 20%, and of these surgeries, approximately 85% are performed due to the presence of initial positive margins. For early stage breast cancer patients, existing guidelines recommend resecting wider negative margins in routine practice, although further surgery and additional tissue removal are often required due to the inherent challenges in achieving negative margins. Currently, surgeons rely primarily on visual and tactile cues to distinguish healthy from malignant tissue, and thus may leave residual disease in the tumor bed. CT, MRI, PET, etc. are readily available imaging modalities typically used to evaluate tumor lesions prior to surgical procedures. Specifically, for breast cancer, mammography with MRI or ultrasound is the current standard of care. Although these conventional imaging modalities have been extremely useful in advancing tumor detection and determining the extent of resection, the need for accurate intraoperative imaging remains one of the most important unmet needs in the management of breast cancer at the time of initial BCS.

[0005] Extensive research is underway to identify new rapid and accurate intraoperative margin assessment tools, some of which are currently undergoing clinical trials. For example, image-guided surgery using fluorescence imaging, among other biophotonic-based technologies, offers surgeons many advantages. Near-infrared (NIR) imaging is an emerging biomedical imaging modality for fluorescence-guided surgery due to its remarkable light absorption, ability to assist real-time visualization, and lack of ionizing radiation. In vivo imaging in the NIR range (700-900 nm) is superior to that in the visible spectrum due to its low scattering, negligible tissue autofluorescence, and relatively high tissue penetration. Indocyanine green (ICG), the first FDA-approved NIR dye, has been used in clinical practice for over 50 years due to its proven safety and feasibility of its use. However, the main drawbacks of using ICG for diagnosis are its very short half-life (2-4 min), its ability to readily bind nonspecifically to plasma proteins, and the lack of a tumor-specific ligand-receptor interaction mechanism as a passive fluorescent dye. Therefore, creating new tumor-targeted versions of ICG could be useful in clinics. Summary of the Invention

[0006] Current cancer treatment involves resection of both the tumor and surrounding normal tissue margins, followed by systemic therapy for early and locally advanced disease. However, due to the inherent challenges associated with achieving negative margins, further surgery and tissue removal are often necessary. Novel methods to optimize surgical resection and confirm tumor-free margins are urgently needed. We developed a unique near-infrared (NIR) fluorescent imaging probe, ICG-p28, composed of the clinically non-toxic tumor-targeting peptide p28 and the FDA-approved NIR dye indocyanine green (ICG). Herein, we analyzed the in vivo kinetics and identified small (≦1 mm) lymph node (LN)-containing cancer cells using intraoperative imaging with ICG-p28 to optimize the clinical practice setting. Xenograft tumors stably expressing iRFP as a tumor marker showed significant colocalization with ICG-p28 but not ICG alone. Image-guided surgery with ICG-p28 reduced residual normalized tumor DNA at the margins by 6.6x10 compared to the control approach (i.e., surgery with ICG or palpation / visual inspection only). 3 It showed a more than fold reduction in tumor recurrence rates (92% specificity) in multiple breast cancer animal models, independent of receptor expression status. ICG-p28 enabled precise identification of tumor cells at the margins to increase the likelihood of complete resection. Our simple and cost-effective approach allows surgeons to intraoperatively identify tumor margins in a real-time 3D manner, providing a new surgical procedure that significantly improves overall outcomes by reducing re-excision rates.

[0007] Other methods, features and / or advantages will become or become apparent upon examination of the following drawings and detailed description, and it is intended that all such additional methods, features and advantages be included within this description and protected by the accompanying claims.

[0008] Brief Description of the Sequence Listing SEQ ID NO:1 is a primer specific for the human-specific Alu sequence (huAlu), and has the sense: 5'-ACG CCT GTA ATC CCA GCA CTT-3' (SEQ ID NO:1).

[0009] SEQ ID NO:2 is a primer specific to the human-specific Alu sequence (huAlu), and the antisense sequence is 5'-TCG CCC AGG CTG GAG TGC A-3' (SEQ ID NO:2).

[0010] SEQ ID NO:3 is a primer specific to the mouse GAPDH genomic DNA sequence (mGAPDH), and its sense is 5'-AGG TCG GTG TGA ACG GAT TTG-3' (SEQ ID NO:3).

[0011] SEQ ID NO: 4 is a primer specific to the mouse GAPDH genomic DNA sequence (mGAPDH), and the antisense sequence is 5'-GGG GTC GTT GAT GGC AAC A-3' (SEQ ID NO: 4).

[0012] SEQ ID NO:5 is p28 from azurin of P. aeruginosa

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0014] [Figure 1A] 1 is a graph of NIR fluorescence of ICG-p28 as a function of concentration. [Figure 1B] 1 is a graph of the signal-to-noise ratio of the NIR signal under various conditions. [Figure 1C] The NIR signal as a function of size is shown. [Figure 1D] 1 is a graph of NIR signal as a function of depth in a gelatin-based phantom. [Figure 1E] FIG. 1 is a schematic diagram of the creation of a gelatin-based phantom.

[0015] [Figure 2A] Real-time imaging with ICG-p28 in a xenograft mouse model. MDA-MB-231 breast cancer cells were injected sc into the mice. White light photographs (top) and snapshot NIR PDE images (bottom; dorsal view, grayscale at 800 nm) are shown. [Figure 2B] Real-time imaging with ICG-p28 in a xenograft mouse model. Mice were injected sc with IOWA-1T breast cancer cells. White light photographs (top) and snapshot NIR PDE images (bottom; dorsal view, grayscale at 800 nm) are shown. [Figure 2C] H&E stained sections of the primary tumor (top) and of the sciatic LN located in the superficial gluteal muscle (bottom; yellow arrowhead in b), confirmed to be tumor positive (magnification 200x (inset 40x)).

[0016] [Figure 3A] Colocalization of ICG-p28 with tumor marker iRFP. ICG-p28-treated MDA-MB-231-iRFP cells were fixed in paraformaldehyde and images were acquired under confocal microscope. Red: tumor marker iRFP (700 nm); Green: ICG-p28 (800 nm); Blue: DAPI (nuclei); Yellow: red + green. [Figure 3B] Colocalization of ICG-p28 with the tumor marker iRFP. Athymic mice bearing MDA-MB-231-iRFP tumors were injected iv with either ICG-p28 or ICG alone at 0.5 mg / kg. 24 hours after injection, representative ex vivo images of tumors and surrounding soft tissues in the ICG-p28 group (top row) and ICG alone group (bottom row) were taken with an Odyssey scanner. The dotted lines in the images indicate the outer edge of the soft tissues. iRFP (red: 700 nm); ICG-p28 or ICG (green: 800 nm); and colocalization (yellow-orange). [Figure 3C]Colocalization of ICG-p28 with the tumor marker iRPF. The coincidence rate of the fluorescent signals in each group [ICG-p28 group (n=10) and ICG only group (n=5)]. **: P<0.01. The results are shown as the mean + SD. [Figure 3D] Colocalization of ICG-p28 with the tumor marker iRFP. Breast cancer cells expressing the NIR fluorescent protein iRFP (700 nm). MDA-MB-231 cells stably expressing the iRFP gene (MDA-MB-231-iRFP) were selected in the presence of puromycin, and the NIR fluorescence from iRFP (grayscale of 700 nm) was confirmed with an Odyssey® scanner.

[0017] [Figure 4A] FIG. 1 is a schematic diagram of intraoperative imaging performed during tumor resection in a human breast cancer xenograft tumor model. [Figure 4B] Representative images of tumor resection sites at the end of the study. [Figure 4C] The mean volume of recurrent tumors in each group (4 weeks after tumor resection) is shown in the graph. Results are presented as mean + SD.

[0018] [Diagram 5]Figure 5A-5E: Image-guided surgery of human breast cancer PDX model. TNBC PDX cells were inoculated into the 4th abdominal fat pad. 24 hours after NIR agent injection (Figure 5A, ICG-p28; Figure 5B, ICG alone), tumors were excised under visualization by PDE imaging unit. White light photographs (left) and snapshot NIR PDE images (right; dorsal view, grayscale at 800 nm) are shown. Yellow arrowhead: tumor. Figure 5C: Signal-to-noise ratio of PDX-bearing mice was recorded with PDE unit and quantitatively analyzed (N=8 per group). ***: P<0.001. Results are shown as mean + SD. Figure 5D-5E: TNBC PDX cells were inoculated into the 4th abdominal fat pad. 24 hours after NIR agent injection (Figure 5D, ICG-RI p28; Figure 5E, ICG-AA3H), tumors were excised under guidance by PDE imaging unit. White light photograph (left) and snapshot NIR PDE image (right; dorsal view, grayscale at 800 nm) are shown. Yellow arrowhead: tumor.

[0019] [Figure 6A] FIG. 1 is a schematic diagram of biopsy collection sites from NSG mice bearing PDX tumors. [Figure 6B] Box plots showing log(huAlu DNA / mGAPDH DNA) values ​​calculated using standard amounts of huAlu DNA and mGAPDH DNA between drugs. *: P<0.05, **: P<0.005. Results are shown as mean ± SD. [Figure 6C] Histological analysis of surgical specimens is shown. Representative images (200x) of H&E staining and staining for the proliferation marker Ki-67 to confirm the presence of negative margins in the specimen obtained from the ICG-p28 group (top). The specimen obtained from the ICG group (bottom) shows the presence of malignant cells. [Figure 6D] 1 shows a graph of the tumor volume in each group after 4 weeks. [Figure 6E] Histological analysis of surgical specimens is shown, representative images (200x) of H&E staining and staining for the proliferation marker Ki-67.

[0020] [Figure 7A]The fold increase in residual tumor DNA in surrounding tissues from the ICG-p28 group in Figure 6B is presented. [Figure 7B] Normality test of the distribution of huAlu / mGAPDH values.

[0021] [Figure 8A] H&E stained sections from the ICG-p28 group. After tumor removal, the surrounding mammary tissue showed atypical hyperplasia (arrowheads). [Figure 8B] H&E stained sections from the ICG-p28 group. Recurrent tumor sections were obtained 4 weeks after image-guided surgery with ICG-p28. Magnification 200x (inset 40x). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0023] As used herein, the term "cell" includes either the singular or plural of this term. The terms "isolated," "purified," or "biologically pure" refer to a material that is substantially or essentially free from components that normally accompany the material as found in its natural state. As used herein, terms such as "heterologous DNA," "heterologous nucleic acid sequence," or "exogenous" refer to a nucleic acid sequence in which at least one of the following is true: (a) a sequence of nucleic acid that is foreign (i.e., not found in nature) to a given host microorganism; (b) the sequence may be found naturally in a given host microorganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acid includes two or more subsequences that are not found in the same relationship to each other in nature.

[0024] "Peptide" and "polypeptide" are used interchangeably herein to refer to a compound comprised of a chain of amino acid residues linked by peptide bonds. An "active portion" of a polypeptide means a peptide that is less than the full-length polypeptide but that retains measurable biological activity and retains biological detection.

[0025] As used herein, the term "tumor" refers to any neoplastic growth, proliferation, or mass of cells, whether benign or malignant (cancerous), either as a primary site lesion or as a metastasis.

[0026] As used herein, a "therapeutically effective amount" refers to an amount of a composition that relieves (to some extent, as judged by a skilled physician) one or more symptoms of a disease or condition in a mammal. Moreover, a "therapeutically effective amount" of a composition means an amount that partially or completely returns any of the physiological or biochemical parameters associated with or causing the disease or condition to normal. One skilled in the art can determine a therapeutically effective amount of a composition when administered intravenously, subcutaneously, intraperitoneally, orally, or by inhalation, etc., to treat or prevent a particular disease condition or disorder. The exact amount of a composition required to be therapeutically effective depends on many factors, such as, for example, the specific activity of the active agent, the delivery device used, the physical properties of the agent, the purpose of administration, in addition to many patient-specific considerations. However, determining a therapeutically effective amount is within the skill of one of ordinary skill in the art upon incorporation of the disclosure set forth herein.

[0027] As used herein, "treat", "treating", "treatment" and the like refer to any action that provides benefit to a patient at risk of or suffering from a disease, including amelioration of symptoms by reducing or suppressing at least one symptom, delaying the progression of the disease, preventing or delaying the onset of the disease, and the like. Treatment also includes partial or complete destruction of undesirable proliferating cells with minimal destructive effects on normal cells. A subject at risk is a subject who has been determined to have a higher than average risk of developing cancer, which can be determined, for example, by family history or detection of a gene that causes a predisposition to developing cancer.

[0028] The term "subject" as used herein refers to mammalian species, including, but not limited to, primates, including monkeys and humans, equines (e.g., horses), canines (e.g., dogs), cats, various farm animals (e.g., ungulates, such as pigs, hogs, goats, sheep, etc.), as well as domesticated pets and animals kept in zoos.

[0029] Where methods and steps described herein indicate particular events occurring in a particular order, those skilled in the art will recognize that the order of certain steps may be modified and that such modifications are in accordance with variations of the invention. Additionally, certain steps may be performed simultaneously in parallel processes where possible, or may be performed sequentially.

[0030] The meanings of the abbreviations are as follows: as is clear from the usage, "C" means Celsius or degrees Celsius, "s" means second(s), "min" means minute(s), "h", "hr" or "hrs" means hour(s), "psi" means pounds per square inch, "nm" means nanometer, "d" means day(s), "μL" or "uL" or "ul" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "mm" means millimeter(s), "nm" means nanometer, "mM" means millimole, "μM" or "uM" means micromole, and "M" means mole. "mmol" means millimole(s), "μmol" or "uMol" means micromole(s), "g" means gram(s), "μg" or "ug" means microgram(s), "ng" means nanogram(s), "PCR" means polymerase chain reaction, "kDa" means kilodalton, "g" means the gravitational constant, "bp" means base pair(s), "kbp" means kilobase pair, "%w / v" means weight / volume percent, "%v / v" means volume / volume percent, "rpm" means revolutions per minute, "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

[0031] overview Tumor-targeted delivery of imaging agents or drugs is one of the major challenges in cancer therapy. Because targeted delivery may improve the usefulness of such agents, various delivery vehicles including small molecules, antibodies, proteins, and peptides have been investigated. Peptides are generally more specific than small molecules due to their high complexity and are relatively inexpensive to produce. In particular, cell-penetrating peptides (CPPs) have been shown to be promising agents for improving the delivery and cellular uptake of diagnostic and therapeutic agents. They also have advantages over larger molecular weight antibodies in terms of synthetic feasibility, flexibility of derivatization, low immunogenicity, and physicochemical parameters. These characteristics suggest that the development of CPPs as carrier molecules is a promising strategy for tumor-targeted delivery.

[0032] Pseudomonas aeruginosa azurin and its derived peptide p28 preferentially enter various cancer cells and induce p53-mediated antiproliferative effects. As a single therapeutic agent, p28 (NSC745104) has been tested in two Phase I clinical trials and granted FDA Orphan Drug and Rare Pediatric Disease designations due to its demonstrated preliminary efficacy in patients with advanced solid tumors and pediatric patients with recurrent and refractory central nervous system (CNS) tumors (NCI and the Pediatric Brain Tumor Consortium) without apparent adverse effects, toxicity, or immunogenicity. Thus, p28 is a potentially ideal CPP that can serve as a tumor-targeting carrier because it preferentially penetrates cancer cells, is highly water-soluble, stable, and clinically shows no significant adverse effects, toxicity, or immunogenicity in humans. Here, we developed a novel non-invasive NIR-based probe, ICG-p28, composed of clinically non-toxic p28 and FDA-approved ICG, and demonstrated that image-guided surgery with ICG-p28 clearly defined margins in a real-time 3D manner, resulting in adequate tumor resection in clinically relevant situations. These findings promise improved outcomes in BCS and can guide the development of simplified and more cost-effective protocols (in terms of overall procedure / operation time).

[0033] A new optical imaging probe, ICG-p28, was characterized with respect to its photophysical properties in vitro, its specific uptake in tumors, and its ability to accurately identify tumor margins in multiple mouse models of human breast cancer. Intraoperative imaging with ICG-p28 was able to identify small (≤1 mm) LN metastases. Image-guided surgery with ICG-p28 improved positive margin identification and tumor recurrence rates in multiple animal models of breast cancer, independent of receptor expression status. In general, fluorescent imaging is a highly beneficial technique for real-time assessment of tumor borders during surgical procedures, offering several advantages, such as the relatively low cost of fluorescent dyes, as well as safety for patients and medical teams, since non-invasive agents are used. Furthermore, optical imaging with contrast enhancement can be used to visualize tumors before resection without altering the surgical field. Due to preferential penetration of ICG-p28, substantial contrast between tumors and surrounding normal tissues was achieved in multiple animal models of breast cancer.

[0034] It is understood that "p28 probe" may refer to any detectably labeled p28 molecule. Preferably, p28 comprises SEQ ID NO: 5, which is at amino acids 50-77 of azurin obtained from P. aeruginosa. It is recognized that any polypeptide comprising SEQ ID NO: 5 or at least 96% identical to p28 can be used as a probe in the methods described herein. That is, if a single amino acid among the 27 amino acids of p28 is changed by substitution with a different amino acid, the new sequence will be 96% identical to p28. Similarly, if a single amino acid is added to the p28 sequence, a sequence 96% identical to p28 is obtained. A "p28 probe" may include conservative amino acid substitutions or unnatural amino acids so as to still fall within the scope of the term "p28 probe". Similarly, it is understood that a "p28 probe" may be modified with any detectably labeled substance, although in this case, preferably, the detectable label is any near-infrared label.

[0035] In some embodiments, compositions of detectably labeled p28 probes are described herein, wherein the detectable label comprises a near-infrared fluorescent molecule. In some embodiments, the near-infrared fluorescent molecule is indocyanine green.

[0036] In some embodiments, a method of detecting cells expressing a tumor marker is described herein. The method includes exposing the cells to a detectably labeled p28 probe and performing an imaging technique to detect the probe. The method is effective in that the probe co-localizes with cells expressing the tumor marker, thus detecting cells expressing the tumor marker. In some embodiments, the detectable label of the p28 probe is a near-infrared fluorescent molecule, and in some embodiments, the molecule is indocyanine green. In some embodiments, the probe exhibits a detectable fluorescent signal at a concentration of about 0.2 pM to about 20 μM.

[0037] In some embodiments, a method of identifying tumor cells in a subject during surgery is described herein. The subject may be suspected of having a tumor or may be a subject diagnosed with having a tumor. The method includes administering a detectably labeled p28 probe to the subject and performing surgery to remove tumor cells from the subject, in which the probe is localized to the tumor cells. During the surgical procedure, an imaging technique is performed that provides real-time images to detect cells labeled by the probe, the cells being cells labeled by the probe. In some embodiments, the detectable label of the p28 probe is a near-infrared fluorescent molecule or is indocyanine green. In some embodiments, the probe is administered to the subject at a concentration of about 50 nM to about 250 nM. In some embodiments, the probe is administered intravenously or subcutaneously. In some embodiments, the period between administration of the probe and performing the imaging technique is about 24 hours. In some embodiments, the real-time imaging technique depicts the margins of the tumor during the surgical procedure. In some embodiments, the method is performed on a subject diagnosed with breast cancer. In some embodiments, the intraoperative imaging technique includes a CCD camera unit that detects near-infrared (NIR) signals at 800 nm. In some embodiments, the probe is localized to tumors that are about 1 mm in size. In some embodiments, subjects are found to have reduced tumor recurrence after surgical removal of tumors when compared to subjects who undergo surgery to remove tumors without additional imaging techniques during the surgical procedure.

[0038] In some embodiments, the present invention describes post-operative evaluation of a subject for residual tumor cells by administering a detectably labeled p28 probe to a subject undergoing surgery to remove a tumor and subjecting the subject to an imaging technique to detect cells labeled by the probe. The imaging technique positively identifies cells expressing the tumor marker, and thus identifies any residual tumor cells. EXAMPLES

[0039] Example 1: Characterization of ICG-p28 in vitro: and in vivo kinetics Since ICG-p28 is a new NIR compound, we characterized its photochemical properties using the PDE neo® imaging unit, an FDA 510(k)-cleared NIR system consisting of a CCD camera unit that detects NIR signals at 800 nm. This PDE system (camera coupled with a small control unit and LCD monitor) has been used for clinical applications including NIR imaging with ICG to identify liver segments and bile ducts, among other physiological systems. The advantage of the PDE unit is its size and portability, which allows the surgeon to hold the camera unit (1.1 lb) and easily obtain real-time NIR images from various angles during surgery. First, we tested the sensitivity of the PDE unit in detecting ICG-p28. ICG-p28 was placed in a 5% intralipid emulsion, which is reported to mimic the light scattering and absorption properties of tissue. Both ICG-p28 and ICG alone showed nearly identical fluorescence quantum yields (ΦF) at concentrations ranging from 0.2 pM to 20 μM in a concentration-dependent manner.

[0040] Referring now to FIG. 1E, a schematic diagram of NIR imaging of a gelatin-based breast phantom and creation of the gelatin-based phantom is described. A tubular inclusion (tumor) containing 200 nM ICG-p28 in agarose was created. Subsequently, 10% molten gelatin in Tris buffer was mixed with 170 μM bovine hemoglobin and 1% (v / v) intralipid emulsion (20%). The gelatin solution was poured into a 50 ml tube. The agarose tumor stimulating inclusion containing ICG-p28 was placed in the center of the warm gelatin-based phantom. The phantom was allowed to solidify overnight at 4° C. Once solidified, the phantom was removed from the 50 ml tube and placed on a flat table. The PDE camera was set above the phantom (a distance of 200 mm was maintained throughout). To determine the depth detection limit, the phantom was sliced ​​in layers 2 mm at a time until the inclusion was reached. At all depths, images were taken in real time to quantitatively measure intensity.

[0041] Further referring to FIG. 1, characterization of ICG-p28 is demonstrated. FIG. 1A, NIR fluorescence from ICG-p28 or ICG in 5% Intralipid emulsion was measured at 3 mW / cm 2The fluorescence quantum yield (ΦF) of ICG-p28 was similar to that of ICG at picomolar to micromolar concentrations. Figure 1B, Signal-to-noise ratios (signal at 800 nm: tumor, noise: surrounding normal tissue, thigh muscle) in MDA-MB-231 tumor-bearing mice were recorded with a PDE unit and quantitatively analyzed in a dose-dependent manner. Prescans were performed before injection of the NIR agent. ***: P<0.001, *: P<0.05, ns: not significant. Figure 1C, Specific NIR signals (800 nm) of tumor-mimicking inclusions of different sizes (1, 2, 3 and 5 mm) containing 200 nM ICG-p28 were measured. Figure 1D, The specific NIR signal (800 nm) of a tumor-mimicking inclusion containing 200 nM ICG-p28 inserted into the phantom at a depth of 0–2 cm was measured. Figure 1D(i), Side view of the phantom. Figure 1D(ii), Top view of the phantom. Red arrow: location of the tumor-mimicking inclusion. PDE camera (3 mW / cm 2 The distance between the phantom and the objective lens was fixed at 200 mm. Results are presented as mean + SD.

[0042] The use of antibodies conjugated to ICG is limited because ICG loses its NIR fluorescence due to the interaction between ICG and the large molecular weight / multiple domain IgG molecule (approximately 150 kDa). In contrast, ICG did not lose its fluorescence when conjugated to p28, likely due to the size of p28 (2.9 kDa, approximately 1 / 50 the size of IgG).

[0043] Preclinical studies of p28 as a single therapeutic agent and the results of two phase I clinical trials identified a therapeutic dose of p28 for treating cancer patients. Because the fluorescence imaging technique depends on the concentration of each contrast agent, the optimal dose range of ICG-p28 in mice bearing MDA-MB-231 human breast cancer tumors was determined in PDE units. Each mouse was injected iv with ICG-p28 or ICG alone at 0.2-1.0 mg / kg (approximately 50-250 nM). Mice were imaged at various time points (6-72 h) and the ratio of NIR signal intensity of tumor tissue to normal tissue (thigh) was calculated. Significant contrast between tumor and surrounding normal tissue was detected 24 h after injection of 0.2-1.0 mg / kg ICG-p28 (Figure 1B). Because higher doses of ICG-p28 (>1.0 mg / kg) required longer times to achieve clear contrast ("washout") due to high background signal, tumor-specific signal was determined to be optimal at concentrations of 0.2-0.5 mg / kg within a practical timescale for clinical use (24 h) (Figure 1B). In contrast, ICG alone at the same dose was rapidly excreted and could not discern the tumor site according to NIR imaging (tumor / normal tissue ratio ≈1) (Figure 1B). This result indicates preferential uptake of p28, resulting in longer retention at the tumor site and clear contrast imaging of the tumor relative to the surrounding tissue.

[0044] As mentioned above, the use of optical techniques at NIR wavelengths for tissue measurements is advantageous as it can avoid the effects of autofluorescence and allow for the measurement of relatively deep tissues. To determine the detection limit for the size and depth of interest, tumor-mimicking inclusions of various sizes containing ICG-p28 (Figure 1C) and phantoms incorporating these inclusions at predefined locations (Figure 1D) were imaged with the PDE unit. Even with the smallest objective lens (1 mm), NIR fluorescence imaging of ICG-p28 at nanomolar concentrations was able to detect NIR-specific signals (Figure 1C) and probe tissue at a depth of approximately one centimeter in intralipid emulsions that mimic tissue scattering properties (Figure 1D).

[0045] Based on these imaging and dosing parameters, we performed real-time PDE imaging of immunocompromised mice harboring two distinct subtypes of human breast cancer: estrogen receptor (ER)-positive breast cancer, which accounts for approximately 80% of all human breast cancers, and triple-negative breast cancer (TNBC; negative for ER, progesterone and Her2 receptor expression), which accounts for 10-15% of all human breast cancers and generally exhibits a worse prognosis than ER-positive breast cancer.

[0046] With reference to Figure 2, real-time imaging with ICG-p28 in a xenograft mouse model is demonstrated. MDA-MB-231 (Figure 2A) or IOWA-1T (Figure 2B) breast cancer cells were injected sc into mice. Once tumors (<10 mm) developed, mice were injected iv with 0.5 mg / kg ICG-p28. 24 hours after ICG-p28 injection, mice were imaged with the PDE system during tumor removal. White light photographs (top) and snapshot NIR PDE images (bottom; dorsal view, grayscale at 800 nm) are shown. The liver (green arrow) showed relatively high signal intensity from excretion of ICG. Red arrow: tumor. Figure 2C shows H&E stained sections of the primary tumor (top) and the sciatic LN (bottom; yellow arrowhead in b) located in the superficial gluteal muscle, confirmed to be tumor positive (magnification 200x (inset 40x)).

[0047] Athymic mice xenografted with sc MDA-MB-231 (TNBC) (Figure 2A) or IOWA-1T (ER+, PR-, Her2-) human breast cancer cells (Figure 2B) were injected with a single iv dose of ICG-p28 at 0.5 mg / kg (~125 nmole / kg). 24 h after ICG-p28 injection, we observed preferential uptake of ICG-p28 by tumors and clear contrast between tumors and surrounding tissues (Figure 2A-B), suggesting that our approach can be applied to a wide range of breast cancer types independent of receptor expression patterns. Notably, during tumor resection, intraoperative NIR imaging revealed a period of concentrated preferential uptake of ICG-p28 in small (≤1 mm) sciatic lymph nodes (LNs) (Figure 2B). Hematoxylin and eosin (H&E) stained sections confirmed the presence of cancer cells within the LNs (Figure 2C), suggesting that ICG-p28 can identify other cancer foci within the breast parenchyma.

[0048] Example 2: ICG-p28-based assessment of tumor margins in the MDA-MB-231-iRFP tumor-bearing model To determine whether ICG-p28 can accurately delineate tumor margins, we generated the MDA-MB-231-iRFP (TNBC) cell line, which stably expresses the NIR fluorescent protein iRFP as a tumor marker, providing an excellent experimental model to evaluate the accuracy of tumor margin detection according to real-time comparison of fluorescence between iRFP and ICG-p28.

[0049] Referring now to FIG. 3A, ICG-p28-treated MDA-MB-231-iRFP cells were fixed in paraformaldehyde and images were acquired under a confocal microscope. Red: tumor marker iRFP (700 nm); Green: ICG-p28 (800 nm); Blue: DAPI (nuclei); Yellow: red + green. In FIG. 3B, athymic mice bearing MDA-MB-231-iRFP tumors were injected iv with either ICG-p28 or ICG alone at 0.5 mg / kg. 24 hours after injection, representative ex vivo images of tumors and surrounding soft tissues in the ICG-p28 group (top row) and ICG alone group (bottom row) were taken with an Odyssey scanner. The dotted lines in the images indicate the outer edge of the soft tissues. iRFP (red: 700 nm); ICG-p28 or ICG (green: 800 nm); and colocalization (yellow-orange). Figure 3C shows the concordance rate of the fluorescent signals in each group [ICG-p28 group (n=10) and ICG only group (n=5)]. **: P<0.01. The results are shown as the mean + SD.

[0050] The 700 nm channel can be used to monitor iRFP-specific fluorescence in cancer cells without affecting the 800 nm fluorescence (and therefore the signal from ICG-p28), and is therefore suitable for detecting tumors generated by these cells in vivo. Confocal images (Figure 3A) and LI-COR Odyssey images (Figure 3D) of MDA-MB-231-iRFP cells showed that intracellular expression of the tumor marker iRFP allows for accurate determination of tumor margins. Athymic mice inoculated with MDA-MB-231-iRFP cells were injected iv with 0.5 mg / kg ICG-p28 or ICG alone. After 24 h, the xenografted tumors and surrounding tissues were removed. The dotted line in Figure 3B indicates the outer edge of the soft tissue. Ex vivo images showed good overlap between the NIR signals (merge) from iRFP (red: 700 nm) and ICG-p28 (green: 800 nm) (Figure 3B). Furthermore, similar to the results presented in Figures 1 and 2, only ICG showed poor tumor retention (Figure 3B). The concordance rates of the 700 and 800 nm NIR signals in the ICG-p28 group and the ICG-only group were 86% and 12%, respectively (Figure 3C). These results indicate that ICG-p28 can preferentially localize to tumor sites and enhance the stability of ICG due to p28 conjugation.

[0051] Example 3: Intraoperative imaging of orthotopic xenografts To evaluate whether real-time image-guided surgery can reduce tumor recurrence, an orthotopic MFP xenograft tumor model was used. With reference to Figure 4, intraoperative imaging of the orthotopic xenograft model is shown. Figure 4A is a schematic diagram of intraoperative imaging performed during tumor resection in a human breast cancer xenograft tumor model. Human breast cancer cells (MDA-MB-231 or IOWA-1T) were inoculated into the fourth abdominal fat pad by sc injection at the base of the nipple. When mice developed tumors, they were injected with PBS, ICG-p28 or ICG at a concentration of 0.5 mg / kg. 24 hours after injection, tumors were excised while visualized with a PDE imaging unit. The exception was the PBS group, whose tumors were identified only by palpation / direct visualization (without imaging guidance). After tumor resection, mice were monitored for an additional 4 weeks to detect tumor recurrence. In Figure 4B, a representative image of the tumor resection site at the end of the study. In Figure 4C, the average volume of recurrent tumors in each group (4 weeks after tumor resection). Results are presented as mean + SD.

[0052] Athymic mice bearing TNBC MDA-MB-231 or ER-positive IOWA-1T tumors were treated with PBS, ICG alone or ICG-p28 (0.5 mg / kg, iv), and NIR fluorescence was monitored with a PDE system during tumor resection surgery, and tumor and surrounding tissues were removed with a safety margin of 2 mm. No obvious adverse events related to the administration of ICG or ICG-p28 were observed during tumor removal, and there were no other complications during the procedure. For control mice treated with PBS, only palpation and direct visualization without fluorescence imaging were used for margin determination. After tumor resection, tumor recurrence resulting from the remaining positive margins at the resection site was evaluated (Figure 4A). Four weeks after tumor resection, recurrence was observed at the surgical site in 31.2% (5 / 16) of mice in the PBS group and 25.0% (4 / 16) of mice in the ICG alone group. In contrast, one of 16 mice (6.3%) in the ICG-p28 group showed tumor recurrence at the resection site. Representative images of the surgical sites for each group are shown (Figure 4B). The mean volumes of recurrent tumors were 944, 197, and 423 mm3 in the PBS, ICG-p28, and ICG groups, respectively (Figure 4C). Taken together, these findings indicate that ICG-p28 improved surgical outcomes by reducing both the incidence of tumor recurrence and the volume of recurrent tumors by aiding in the accurate identification of tumor margins.

[0053] Example 4: Image-guided tumor resection in PDX tumor models

[0054] PDX models are widely used in cancer research due to their distinct advantages in preserving the biological properties of the original human tumor. Given the reduced tumor recurrence observed in orthotopic xenograft models treated with ICG-p28-induced ablation (Figure 4C), we utilized PDX models and primary TNBC fragments (stage IV) to further evaluate ICG-p28 in a more clinically relevant model. Furthermore, to examine whether this result is due to a specific motif of p28, we included an additional control peptide conjugated to ICG. Similar to p28, an annexin-derived non-cationic CPP called AA3H was previously isolated and shown to penetrate cancer cells. The other peptide was retro-inverso p28 (RI p28). RI modification is often used as an approach to create peptidomimetics that represent isomers of the parent peptide (p28) with reversed sequence orientation and reversed chirality of each amino acid residue.

[0055] In use, image-guided surgery of a human breast cancer PDX model is performed as shown in Figure 5. TNBC PDX cells were inoculated into the 4th abdominal fat pad. 24 hours after NIR agent injection (Figure 5A, ICG-p28; Figure 5B, ICG alone), tumors were excised under visualization with a PDE imaging unit. White light photographs (left) and snapshot NIR PDE images (right; dorsal view, grayscale at 800 nm) are shown. Yellow arrowhead: tumor. In Figure 5C, the signal-to-noise ratio of PDX-bearing mice was recorded with the PDE unit and quantitatively analyzed (N=8 per group). ***: P<0.001. Results are shown as mean + SD.

[0056] Under anesthesia, tumors and surrounding mammary tissues were removed with a 2 mm safety margin under real-time guidance by PDE imaging in mice injected with ICG-p28 (Figure 5A), ICG alone (Figure 5B) or control agents (PBS, ICG-RI-p28 Figure 5D or ICG-AA3H; Figure 5E). ICG-p28 provided significantly greater contrast enhancement and more easily identified tumors than control agents. These results indicate that the tumor targeting ability of ICG-p28 is due to the specific motifs of p28.

[0057] Example 5: Detection of residual cancer cells in tumor margins by Alu sequencing

[0058] Referring now to FIG. 6, the detection of residual cancer cells in the tumor margins is described. FIG. 6A shows a schematic of the biopsy collection sites from NSG mice bearing PDX tumors. FIG. 6B shows that genomic DNA was extracted from each specimen and subjected to PCR assay. Box plots show the values ​​of log(huAlu DNA / mGAPDH DNA) calculated using standard amounts of huAlu DNA and mGAPDH DNA between drugs. *: P<0.05, **: P<0.005. Results are shown as mean ± SD. In FIG. 6C, histological analysis of surgical specimens is shown. Representative images (200x) of H&E staining and staining of the proliferation marker Ki-67 to confirm the presence of negative margins in specimens obtained from the ICG-p28 group (top). Specimens obtained from the ICG group (bottom) show the presence of malignant cells. In FIG. 6D, tumor volumes were determined for each group 4 weeks after surgical resection. FIG. 6E provides representative images (200x) of H&E staining and staining for the proliferative marker Ki-67, confirming the presence of positive margins in specimens obtained from the indicated groups.

[0059] To quantitatively determine the effectiveness of margin identification by our imaging approach, a PCR-based assay was used to detect residual human cancer cells in the PDX model described above (Figure 5). During image-guided surgery, tumor and surrounding breast tissues were removed in real time at the 6 o'clock and 12 o'clock positions with a 2 mm safety margin under the guidance of the PDE imaging system (Figure 6A). These samples were collected for huAlu PCR as well as conventional histological analysis. In quantitative analysis using huAlu and the housekeeping gene mGAPDH, image-guided surgery with ICG-p28 showed significantly fewer residual cancer cells in the surrounding tissues [log(huAlu DNA / mGAPDH DNA)=-5.2] than surgery with other agents [ICG alone, ICG-AA3H, ICG-RI p28 or PBS: -0.9, -1.9, -0.5 and -2.1, respectively] (Figure 6B, Figure 7).

[0060] Specifically referring to FIG. 7, the results of normality testing of huAlu / mGAPDH values ​​are shown. In FIG. 7A, the fold increase in residual tumor DNA in surrounding tissues from the ICG-p28 group was calculated based on the data shown in FIG. 6B (#). Image-guided surgery with ICG-p28 resulted in a 6.6×10 increase in residual normalized tumor DNA at the margin compared to surgery with a control agent. 3 This resulted in an average fold decrease in huAlu / mGAPDH levels. In Figure 7B, a normality test was performed to assess the normality of the distribution of log-transformed huAlu / mGAPDH values ​​using the equation Y = log(huAlu / mGAPDH). The QQ plot shows a linear relationship, indicating that the transformed data can reasonably be treated as normally distributed.

[0061] The findings suggest that image-guided surgery with ICG-p28 reduced residual normalized tumor DNA at the margins by 6.6x10 compared with control surgery. 3The results showed that ICG-p28 produced a mean fold reduction in tumor size and accurately differentiated tumor margins better than the other agents. The coefficient of variation (CV) also indicated that ICG-p28 was superior to other peptides / agents as an intraoperative probe since it showed a relatively small variance as shown in Table 1.

[0062] [Table 1] The coefficient of variation (CV) was calculated, which indicates the variability defined by the standard deviation around the mean. For log-normal data, the formula is

number

[0063] We next performed conventional histological analysis using H&E and proliferation marker Ki67 staining on the set of tumor margin samples used in the Alu PCR assay. The results of these studies revealed that image-guided surgery with ICG-p28 could result in clear negative margins at the edges of breast tissue (Figure 6C). In the ICG-p28 group, one out of 15 breast tissue samples showed atypical hyperplasia, whereas the positive margin rates in the PBS, ICG alone, ICG-RI p28 and ICG-AA3H groups were 19, 19, 31 and 38%, respectively (Figure 6C, Figure 6E). The histological data supported the results obtained from the Alu PCR assay, indicating that image-guided surgery with ICG-p28 improved the ability to accurately identify tumor margins.

[0064] Since tumor recurrence rate is a clinically significant indicator of positive margins, we determined the tumor recurrence rate in the PDX tumor model. Four weeks after image-guided surgery, the tumor recurrence rates at the surgical site were 25.0%, 50.0%, 37.5%, and 25.0% in the PBS, ICG alone, ICG-RI p28, and ICG-AA3H groups, respectively. In the ICG-p28 group, one of eight mice with atypical hyperplasia showed recurrence during tumor resection (Figure 8). The mean volume of recurrent tumors was 2,151, 1,952, 403, 1,152, and 2,447 mm in the PBS, ICG alone, ICG-p28, ICG-RI p28, and ICG-AA3H groups, respectively. 3 (Figure 6D). The mean volume of recurrent tumors in the ICG-p28 group was the smallest of all groups tested. ICG-p28 provided greater contrast enhancement and more easily identified tumors than the control agent (Figure 5C), which provided lower contrast and made it difficult to completely remove the tumors. These findings suggest that image-guided surgery with ICG-p28 can accurately identify tumor margins, resulting in a reduction in tumor recurrence.

[0065] We found that tumor-specific signal was optimal at ICG-p28 concentrations of 0.2-0.5 mg / kg within a time scale practical for clinical use (24 h). With imaging and dosing parameters, ICG-p28 provided significant contrast (TBR ~4-fold) in multiple breast cancer animal models. It is generally accepted that a TBR >3.0-fold is sufficient to provide adequate intraoperative contrast in studies on image-guided surgery and is essential for successful clinical translation. In contrast, ICG alone was rapidly excreted as expected and tumor sites were indistinguishable on associated NIR images (TBR ~1.1). This observation also confirmed that the preferential uptake of the new compound was due to the presence of the p28 motif, resulting in longer retention at the tumor site and precise contrast imaging between tumor and surrounding tissues. Although the molecular weight, pI, charge, hydrophobicity and amino acid composition of RI p28 were exactly the same as those of p28, the overall penetration of RI p28 was significantly reduced and did not show preferential penetration (Figure 6). This finding suggests that the preferential entry of p28 is amino acid sequence-specific / stereochemically specific. Currently, various types of NIR imaging probes are used for various cancers such as glioma and lung, pancreatic, colorectal, prostate and breast cancer. For the latter, a series of investigational fluorescent agents such as bevacizumab-IRDye800 (vascular endothelial growth factor A), EC17 fluorescent dye (folate receptor alpha), cathepsin-activatable fluorescent agent LUM015 (cathepsin protease) and tozleristide (matrix metalloprotease) have been validated for use in image-guided surgery. However, most of these imaging agents are preclinical stage NIR dyes and use antibodies for their targeted delivery (e.g., anti-Her2 antibody). In comparison, the use of p28 has several advantages: i) unlike the use of antibodies, chemical conjugation of p28 to ICG does not alter ICG fluorescence (Figure 1), ii) p28 can target a broad range of breast cancers independent of receptor status (Figures 2, 5, 6), and iii) ICG-p28 is composed of a clinically non-toxic tumor-targeting p28 peptide with an FDA-approved NIR ICG dye.

[0066] Image-guided surgery with ICG-p28 in the PDX model resulted in tumor recurrence in one of eight mice (Figure 6D). However, Alu PCR assays and histological analysis did not reveal residual tumors in MFP specimens obtained at either the 6 o'clock or 12 o'clock positions. There are two possible reasons for the occurrence of this phenotype. One possibility is that cancer cells were not captured by the histological analysis. The specimens were determined to be tumor-negative by histology and Alu analysis but may have contained a few tumor cells that were otherwise missed, indicating the possibility of microinvasive breast tumors less than 1 mm in size. Lesions of this size cannot be seen without a microscope and remain unresected in the tumor bed. In this case, additional approaches (e.g., image-guided surgery with ICG-p28 in combination with other therapeutic approaches such as radiation or chemotherapy) should be further considered to manage these microlesions. Another possibility is that the atypical hyperplasia captured in the histological sections (Figure 8) may have progressed to breast cancer during the 4-week observation period. Although atypical hyperplasia is not cancer, it may transition to invasive / non-invasive breast cancer, possibly due to the characteristics of PDX models. Nevertheless, considering that only one mouse in the ICG-p28 group showed tumor recurrence and the volume of the recurrent tumor was smaller than that of tumors from mice in the other groups, ICG-p28-guided surgery appears to be well-served in accurately identifying tumor margins and significantly reducing the rate of tumor recurrence. Overall, image-guided surgery with ICG-p28 in multiple subtypes of breast cancer (Figure 4, Figure 6) was able to effectively treat tumors up to 300 mm 3 surgery with ICG alone or without intraoperative imaging (PBS) resulted in a mean recurrent tumor volume of 1,188 mm 3 (P = 0.017) and 1,289 mm 3(P=0.032). Moreover, the tumor recurrence rate in the ICG-p28 group was 8% (specificity 92%, N=2 / 24), whereas the tumor recurrence rates in the ICG and PBS groups were 33% (N=8 / 24) and 29% (N=7 / 24), respectively. These results in multiple breast cancer animal models suggest that ICG-p28 improved surgical outcomes by reducing both the incidence of tumor recurrence and the volume of recurrent tumors through the identification of clear margins. Thus, the versatility of ICG-p28 could potentially reduce the risk of recurrence and reoperation in a wide range of breast cancer types regardless of receptor expression status, minimize damage to healthy tissue and medical costs, improve postoperative quality of life, and increase patient survival.

[0067] While many imaging modalities such as MRI, X-ray, CT, PET and ultrasound play a considerable role in preoperative staging and intraoperative planning, fluorescence imaging can be used during intraoperative surgical examination and practice due to its superior resolution and sensitivity. Accurate identification of tumor margins is a primary objective of effective surgical treatment of breast cancer as well as many other types of solid tumors. Insufficient positive margin identification occurs frequently in approximately 5% of lung and kidney tumor resections, approximately 20% of breast, prostate and rectal cancer resections, and up to 40-60% of vulvar and oral cancer resections. Our real-time intraoperative imaging approach may positively impact the outcomes (e.g., morbidity, quality of life and costs) of surgical treatment of many types of solid tumors.

[0068] method Peptide synthesis and ICG conjugation

[0069] The peptides, p28, retro-inverso p28 (RI p28) and AA3H 22, were synthesized by CS Bio, Inc. with >95% purity and mass balance. The peptides were dissolved in PBS buffer at pH 7 and reacted with ICG-maleimide in anhydrous DMSO for 3 h at room temperature in the dark. The reactions were dialyzed against PBS for 24 h at 4° C. (MW 2,000 cutoff, Pierce) and purified by filtration on Sephadex G-25 (GE Healthcare). The final products were identified by HPLC and MALDI mass spectrometry.

[0070] ICG-p28 characterization PDE neo® (Photodynamic Eye; Mitaka USA, UT) is an FDA 510(k) cleared NIR camera consisting of a charge-coupled device (CCD) camera unit that detects NIR signals at 800 nm. In general, the PDE unit (camera and LCD monitor connected to a small control unit) has been used for NIR imaging clinical applications with ICG to identify liver segments and bile ducts. ICG and ICG-p28 in 20% intralipid emulsion (Sigma; St. Louis, MO), which is reported to mimic the light scattering and absorption properties of tissue, were recorded by the PDE camera unit. Various concentrations of ICG-p28 (0.2 pM to 20 μM) and ICG were recorded at 3 mW / cm. 2 The excitation was monitored at 100 Hz.

[0071] Construction of breast tissue-mimicking phantoms

[0072] Optical techniques at NIR wavelengths for tissue measurements are advantageous as they can avoid the effects of autofluorescence and allow for measurements of relatively deep tissues. To determine the depth detection limit, tumor-mimicking inclusions containing ICG-p28 were incorporated into tissue phantoms at predetermined locations and imaged with the PDE unit. A previously published procedure for creating tissue phantoms was modified as shown in Figure 1E. Briefly, 10% gelatin powder (Spectrum; New Brunswick, NJ) was dissolved in 50 mM Tris buffer containing 150 mM NaCl at pH 7.4. The gelatin was dissolved by stirring at 50 °C and subsequently cooled to 37 °C. Bovine hemoglobin and intralipid (20%; Sigma) were then added. To determine the maximum penetration depth of the NIR fluorescent signal, inclusions containing 200 nM ICG-p28 were placed in the phantom tissue. The phantom tissue was subsequently sectioned in layers 2 mm at a time toward the inclusions. Real-time imaging was performed intraoperatively at all depths using the PDE neo system. The distance between the PDE camera and the phantom was fixed at 200 mm. The intensity of the images acquired using the PDE neo system was quantitatively evaluated. Mean ± SD values ​​were calculated.

[0073] cell culture Human breast cancer cell lines MDA-MB-231 and IOWA-1T were obtained from the American Type Culture Collection (Manassas, VA). MDA-MB-231-iRFP cells were established and characterized in our laboratory. MDA-MB-231 cells were transfected with piRFP plasmid with Lipofectamine 2000 (Invitrogen). Transfectants stably expressing the iRFP gene were selected in the presence of 0.5 μg / ml puromycin. They were cultured in MEME medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified incubator containing 5% CO2. Single generation patient-derived xenograft (PDX) cells of triple-negative breast cancer cells (TM00090) were obtained from Jackson Laboratory.

[0074] Animal models All animal studies were performed under protocols approved by the University of Illinois at Chicago. Tumor models were performed at 1.0x10 6 MDA-MB-231, IOWA-1T or MDA-MB-231-iRFP cells were established by subcutaneous (sc) implantation into the right dorsum (kinetic analysis) and mammary fat pad (MFP; qualitative margin analysis) of 4-5 week old female atimic mice (Jackson Laboratory). For additional quantitative margin analysis, 4-5 week old female NOD / Scid / IL2Rγ null Tumor models were established by implanting triple-negative PDX cells (stage IV) into the MFP of (NSG) mice (Jackson Laboratory).

[0075] In vivo kinetic analysis of ICG-p28 MDA-MB-231 cells were injected sc into 4-5 week old female athymic mice. ICG-p28 or ICG alone was injected intravenously (iv) (tail vein) at 0.2, 0.5 or 1 mg / kg-bw. Animals were imaged 6-72 hours after iv administration using an Odyssey scanner (LI-COR Biosciences). Tumor background ratios (TBR) were determined using ImageJ software (National Institutes of Health), where regions of interest (ROIs) were drawn over the entire tumor and background ROIs were drawn over adjacent tissues. ROIs were manually generated around each tumor (800 nm channel).

[0076] Tumor margin assessment MDA-MB-231-iRFP cells (1.0 x 10 in 0.1 ml PBS 6 The mice were inoculated with 100 μg / mL of 10 ... 3Once tumors had developed, mice were injected iv with ICG-p28 or ICG at 0.5 mg / kg-bw. 24 hours after injection, tumors and surrounding tissues were excised. Specimens were imaged ex vivo using an Odyssey scanner with a 700 nm channel for iRFP and an 800 nm channel for ICG-p28 or ICG alone. Area measurements were performed using ImageJ. Mean + SD values ​​were calculated.

[0077] Quantitative assessment of ICG-p28 for determining tumor margins and following tumor recurrence NSG mice are of similar size (200–300 mm 3 Once PDX tumors of 1000-2000 mice had developed, mice were randomly divided into five groups: ICG-p28, ICG alone, ICG-AA3H peptide, ICG-RI p28, and PBS. Each drug was injected i.v. (tail vein) with the corresponding compound equivalent to 0.5 mg / kg-bw of ICG-p28. 24 hours after injection, all surgical procedures were started under sterile conditions. After anesthesia administration, NIR fluorescence was monitored throughout the entire surgical procedure using a PDE imaging system. Under the guidance of the PDE imaging system, tumors and surrounding mammary tissues were removed in real time at the 6 o'clock and 12 o'clock positions with a safety margin of 2 mm. For the PBS group, malignant tissues identified via visual and tactile cues were resected under white light illumination, whereas for the ICG, ICG-p28, ICG-AA3H, and ICG-RI p28 groups, only tissues showing contrast enhancement were resected. Images of the fluorescent signal were displayed on an adjacent monitor, and all ICG-positive tissue foci (suspected as tumor tissue) were excised. After tumor excision, the skin incision was closed using nylon sutures, and the mice were returned to their cages. After tumor excision, the mice were maintained in their cages for an additional 4 weeks and monitored for tumor recurrence. Tumor recurrence was monitored by palpation of the surgical area. Four weeks after surgery, the mice were evaluated to determine whether any of the tumors had recurred due to remaining positive margins at the excision site.

[0078] Detecting residual cancer cells in tumor margins by Alu sequencing Quantitative analysis using human-specific Alu sequences (huAlu) was performed on samples collected from the margins. Because Alu repeat DNA sequences are specific to human cells, human-derived tumors implanted in mice can be identified by detecting the presence of huAlu. We standardized the quantitative detection of huAlu in resected tumors and surrounding margin tissues according to previously published methods. Human tumor cells in five groups (ICG-p28, ICG alone, ICG-AA3H, ICG-RI p28, and PBS; 8 mice per group) were identified based on the quantitative detection of huAlu present in the mice. Genomic DNA was extracted from the collected tissues using the DNeasy Blood&Tissue kit (Qiagen). To identify human cells in mouse tissues, huAlu repeats present in extracted genomic DNA were amplified by real-time PCR (5 ng of genomic DNA, 0.5 μM of each primer, and PowerUp SYBR Green Master Mix; Life Technologies, USA) using primers specific for huAlu (sense: 5′-ACG CCT GTA ATC CCA GCA CTT-3′ (SEQ ID NO: 1); and antisense: 5′-TCG CCC AGG CTG GAG TGC A-3′ (SEQ ID NO: 2). Each reaction was performed in a final volume of 10 μl under the following conditions: polymerase activation at 50°C for 2 min and 95°C for 2 min, followed by 30 cycles of 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s. Quantitative measurements of amplifiable mouse DNA were performed by amplifying the mouse GAPDH genomic DNA sequence (mGAPDH) with mGAPDH-specific primers (sense: 5′-AGG TCG GTG TGA ACG GAT TTG-3' (SEQ ID NO: 3) antisense: 5'-GGG GTC GTT GAT GGC AAC A-3' (SEQ ID NO: 4). Fluorescence emitted by the reporter dye was detected and the threshold cycle (Ct) was recorded as a quantitative measure of the amount of PCR product in each sample.The amounts of huAlu DNA and mGAPDH DNA in the margins were calculated by comparison to the standard curves, and the calculated amounts of huAlu DNA were normalized to the relative amounts of mGAPDH. Mean ± SD values ​​were calculated.

[0079] Histological analysis Resected tissues were placed in containers prefilled with buffered 3.7% formalin (Anatech) and fixed for 24 hours. After fixation, the formalin was replaced with 70% ethanol. Samples were paraffin embedded and blocks were cut into 4 μm thick sections and mounted on slides. H&E- and Ki-67-stained slides were analyzed by a pathologist blinded to the experimental groups and surgical resection results. The pathologist was responsible for determining the presence of tumor, margin status, and general characteristics of the resected tissues.

[0080] statistical analysis Data processing and statistical analysis were performed using GraphPad Prism ver.8 (GraphPad Software) and R version 4.0.5 (The R Foundation for Statistical Computing). Friedman's test and F test were used for recurrent tumor volume data and Alu sequences, respectively.

[0081] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used in carrying out the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. 1. A method for intraoperatively identifying tumor cells in a subject, comprising: administering to a subject a detectably labeled p28 probe; performing surgery to remove tumor cells from the subject; Including, The probe is localized in tumor cells; During the surgical procedure, an imaging technique that provides real-time images is performed to detect cells labeled by the probe, which cells represent tumor cells. method.

2. The method of claim 1 , wherein the detectable label of the p28 probe comprises a near-infrared fluorescent molecule.

3. The method of claim 2 , wherein the near-infrared fluorescent molecule is indocyanine green.

4. The method of claim 1, wherein the probe is administered to the subject at a concentration of about 50 nM to about 250 nM.

5. The method of claim 1 , wherein the probe is administered intravenously or subcutaneously.

6. 10. The method of claim 1, wherein the period between administration of the probe and performance of the imaging technique is about 24 hours.

7. The method of claim 1 , wherein the imaging technique depicts tumor margins.

8. 10. The method of claim 1, wherein the subject has been diagnosed with breast cancer.

9. The method of claim 1 , wherein the imaging technology comprises a CCD camera unit detecting near-infrared (NIR) signals at 800 nm.

10. 10. The method of claim 1, wherein the probe is localized to a tumor approximately 1 mm in size.

11. 10. The method of claim 1, wherein the subject has reduced tumor recurrence following surgical removal of the tumor when compared to a subject undergoing surgery to remove the tumor without additional imaging techniques during the surgical procedure.

12. further comprising post-operatively evaluating the subject for residual tumor cells; administering to a subject undergoing surgery to remove a tumor a detectably labeled p28 probe; applying an imaging technique to the subject to detect cells labeled by the probe; Including, Imaging techniques positively identify cells expressing tumor markers, thereby identifying any remaining tumor cells; The method of claim 1.

13. 1. A method for detecting cells expressing a tumor marker, comprising: exposing the cells to a detectably labeled p28 probe; performing an imaging technique to detect the probe; Including, The probe co-localizes with cells expressing the tumor marker, thereby detecting cells expressing the tumor marker; method.

14. The method of claim 13 , wherein the detectable label of the p28 probe comprises a near-infrared fluorescent molecule.

15. 15. The method of claim 14, wherein the near infrared fluorescent molecule is indocyanine green.

16. The method of claim 13, wherein the probe exhibits a detectable fluorescent signal at a concentration of about 0.2 pM to about 20 μM.

17. A composition comprising a detectably labeled p28 probe, wherein the detectable label comprises a near-infrared fluorescent molecule.

18. 18. The composition of claim 17, wherein the near infrared fluorescent molecule is indocyanine green.

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