Expression prevalence and dynamics of GPCR somatostatin receptors 2 and 3 as cancer biomarkers beyond NET: a paired immunohistochemistry approach.

Somatostatin receptors are clinically validated GPCR biomarkers for diagnosis and treatment of various neuroendocrine tumors (NET). Among the five somatostatin receptors, SST2 and SST3 are associated with apoptosis and cell cycle arrest, making these receptor subtypes better differentiated targets in precision oncology. In this study we performed immunohistochemistry of paired tissue microarrays containing 1125 cores, representing 43 tumor types, each stained for SST2 and SST3. A 12-point immunoreactive scoring (IRS) range was used for interpretation of the staining results. We analyzed the results twice, using the conventional positivity IRS cutoffs ≥ 3 and more stringent ≥ 6. Evaluation of receptors expression dynamics was performed for tumor-nodes-metastases (TNM) defined subgroups (ovarian and hepatocellular adenocarcinomas) as a function of their tumor stage. Our results indicate that two-thirds of tested cores exhibit clinically significant expression of at least SST2 or SST3 (IRS ≥ 6). The expression prevalence of both receptors tends to decline with tumor progression. However, an unexpected upregulation of both SST2 and SST3 reemerged in metastases suggesting conserved receptors genetic potential during tumor life cycle. We suggest that SST2 and SST3 should be further explored as potential biomarkers and therapeutic tools for maximizing the efficiency of somatostatin-based precision oncology of solid tumors beyond NET.

In vivo magnetic resonance imaging of pancreatic tumors using iron oxide nanoworms targeted with PTR86 peptide.

To cut the high mortality rate of malignant disease such as pancreatic cancer, development of newly diagnostic probes for early stage detection of tumor lesions is required. Multimodal imaging nanoprobes allowing targeted and real time functional/anatomical imaging of tumors meet the demands. For this purpose, a MRI/optical dual-modality probe based on biodegradable magnetic iron oxide nanoworms has been developed. The cross-linked surface of nanoworms were anchored to fluorescent dyes and to FITC.PTR86; a novel synthetic peptide with high affinity towards somatostatin receptors. Combination of various in vitro techniques including Prussian blue staining, fluorescent microscopy and fluorescence activated cell sorting (FACS) have been performed to explore the interaction of developed nanoprobe with pancreatic tumor cell lines. Together with in vivo studies in a xenograft mouse model of human pancreatic adenocarcinoma and ex vivo investigations, the results show the efficient imaging and targeting of pancreatic tumors by our newly developed nanosystem using both MRI and optical imaging modalities.

Diagnostic targeting of colon cancer using a novel fluorescent somatostatin conjugate in a mouse xenograft model.

Colorectal carcinoma is one of the more prevalent, highly malignant human tumors, occurring in about 7% of the population. However, if diagnosed and treated in its early stages, colon cancer is curable. In our study, we used a mouse xenograft model to investigate the capability of a fluorescent conjugate of a novel synthetic somatostatin (SST) analog to improve detection of human colorectal tumors that are characterized by over-expressed SST receptors. Human HT-29 colon carcinomas were induced in nude mice. After administration of the fluorescent SST conjugate, in vivo low- and high-magnification fluorescence microscopy, as well as high-resolution spectrally resolved imaging were performed, and the time-dependent biodistribution was determined quantitatively (using fiber-optic spectroscopy). Administration of the conjugate (at concentrations of 6 mg/kg body weight) enabled targeting small (1-5 mm diameter) tumors with high sensitivity and selectivity. Toxicity studies at dosages up to 1,000 mg/kg body weight did not reveal any drug related abnormalities. In conclusion, the SST conjugate significantly enhanced the detection of HT-29 colon tumors by fluorescence imaging because of a 5- to 8-fold increase in the contrast between malignant and normal tissues.

Targeting small-cell lung cancer with novel fluorescent analogs of somatostatin.

Early, accurate detection of small-cell lung cancer (SCLC), before it becomes systemic, is essential for successful treatment. Fluorescence-based imaging provides safe, sensitive detection of malignancies. Targeted delivery of fluorophores increases sensitivity of endoscopic imaging. We synthesized novel somatostatin analogs, based on backbone cyclic peptides, and conjugated them with fluorescent agents. Nineteen conjugates differing in core peptide, length of alkyl linker and fluorescence moiety (rhodamine and fluorescein) were tested in vitro, using a receptor binding assay, and nine of the more promising conjugates were tested in vivo by fiber-optic spectrofluorimetry and quantitative spectral imaging, on an H69 human SCLC tumor mouse xenograft model. The lead compound showed exceptional tumor/normal tissue ratios, ranging from 9 to 90, and has potential for targeting SCLC overexpressing somatostatin receptors.

Improved pharmacokinetics, biodistribution and necrosis in vivo using a new near infra-red photosensitizer: tetrahydroporphyrin tetratosylat.

The search for better photosensitizers for photodynamic therapy of malignancies has led to the investigation of a new water-soluble, positively charged, and chemical stable tetrahydroporphyrin tetratosylat (THPTS) with a strong absorption at 760.5 nm, belonging to the bacteriochlorophyll family. THPTS undergoes a rapid uptake by human choroidal melanoma (CM) cells with a weak dark toxicity after a 24-h incubation (LD10 = 150 microM, LD50 = 6.0 mM). In response to laser light at 760+/-3 nm and doses of 10, 15 and 30 J/cm2, around 71%, 76%, and 92% of the CM cells were killed, respectively. Studies of pharmacokinetics and biodistribution in vivo (living mice) and ex vivo (excised organs) were made in a Balb/c mice bearing subcutaneously inoculated C26 colon carcinoma using fiber-optic spectrofluorimetry (FOS). Tumours were irradiated 3 h after intraperitoneal (i.p.) injection of 5.0 mg/kg THPTS with an incoherent light source at 750+/-20 nm and an intensity of 100 mW/cm2 and fluences of 60, 90 and 120 J/cm2. THPTS demonstrated preferential accumulation in C26 colon carcinoma in comparison with most normal tissues except kidneys. For the tissues of liver, colon, muscle, and spleen the tumour/normal tissue ratio (TNTR) ranged from 8.0 to 50. After irradiation with 120 J/cm2 the depth of tumour necrosis reached 15 mm. Histological examination of the tumour samples 24 h after THPTS-PDT, revealed severe stasis in the blood vessels and coagulative necrosis. These results suggest that THPTS-PDT may be of particular importance in the treatment of accessible malignancies.

Monitoring PDT-induced damage using spectrally resolved reflectance imaging of tissue oxygenation.

Photodynamic therapy (PDT) with chlorin e6 (Chl) was monitored in vivo using vital microscopy and Fourier transform spectral imaging (FT-SI). Mammary C26 colon carcinoma, implanted intradermally in a mouse, was irradiated at 650 nm with various radiant exposures, 3 h after administration of 5 mg/kg Chl. The photodynamic response (PDR) in the skin flap with tumor was expressed as microcirculation disturbances (thrombi formation, multiple embolizations, arteriolar occlusion and venous stasis) and, dependent on the radiant exposure, was transient or permanent. These biological manifestations were accompanied by a change in hemoglobin (Hb)/oxyhemoglobin (HbO2) absorption spectra obtained in vivo by FT-SI. False-color mapping of hemoglobin oxygen saturation (OS) visualized the alteration of tissue oxygenation. The results demonstrate, for the first time, that FT-SI can serve as a sensitive non-invasive tool for OS monitoring of PDT effects.

High-b-value diffusion-weighted MR imaging for pretreatment prediction and early monitoring of tumor response to therapy in mice.

PURPOSE: To evaluate the use of diffusion-weighted magnetic resonance (MR) imaging with standard and high b values for pretreatment prediction and early detection of tumor response to various antineoplastic therapies in an animal model. MATERIALS AND METHODS: Mice bearing C26 colon carcinoma tumors were treated with doxorubicin (n = 25) and with aminolevulinic acid-based photodynamic therapy (n = 23). Fourteen mice served as controls. Conventional T2-weighted fast spin-echo and diffusion-weighted MR images were acquired once before therapy and at 6, 24, and 48 hours after treatment. Pretreatment and early (1-2 days) posttreatment water diffusion parameters were calculated and compared with later changes in tumor volumes measured on conventional MR images by using the Pearson correlation test. RESULTS: In chemotherapy-treated tumors, a significant correlation (P <.002, r = 0.6) was observed between diffusion parameters that reflected tumor viability, measured prior to treatment, and changes in tumor volumes after therapy. This correlation implies that tumors with high pretreatment viability will respond better to chemotherapy than more necrotic tumors. In tumors treated with photodynamic therapy, no such correlation was found. Changes observed in water diffusion 1-2 days after treatment significantly correlated with later tumor growth rate for both therapies (P <.002, r = 0.54 for photodynamic therapy; P <.0003, r = 0.61 for chemotherapy). CONCLUSION: High-b-value diffusion-weighted MR imaging has potential use for the early detection of response to therapy and for predicting treatment outcome prior to initiation of chemotherapy.

Wavelength-dependent properties of photodynamic therapy using hypericin in vitro and in an animal model.

Wavelength effects in photodynamic therapy (PDT) with hypericin (HY) were examined in a C26 colon carcinoma model both in vitro and in vivo. Irradiation of HY-sensitized cells in vitro with either 550 or 590 nm caused the loss of cell viability in a drug- and light-dose-dependent manner. The calculated ratio of HY-based PDT (HY-PDT) efficiencies at these two wavelengths was found to correlate with the numerical ratio of absorbed photons at each wavelength. In vivo irradiation of C26-derived tumors, 6 h after intraperitoneal administration of HY (5 mg/kg), caused extensive vascular damage and tumor necrosis. The depth of tumor necrosis (d) was more pronounced at 590 than at 550 nm and increased when the light dose was raised from 60 to 120 J/cm2. The maximal depths of tumor necrosis (at 120 J/cm2) were 7.5+/-1.5 mm at 550 nm and 9.9+/-0.8 mm at 590 nm. Both values are rather high in view of the limited penetration of green-yellow light into the tissue. Moreover, the depth ratio, d590/d550 = 1.3 (P < 0.001), is smaller than expected considering the 2.2-fold lower HY absorbance and the 1.7-fold lower tissue penetration of radiation at 550 than at 590 nm. This finding indicates that in vivo the depth at which HY-PDT elicits tumor necrosis is not only determined by photophysical considerations (light penetration, number of absorbed photons) but is also influenced significantly by other mechanisms such as vascular effects. Therefore, despite the relatively short-wavelength peaks of absorption, our observations suggest that HY is an effective photodynamic agent that can be useful in the treatment of tumors with depths in the range of 1 cm.