Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins

Purpose

To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features.

Materials and Methods

Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma.

Results

Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach.

Conclusion

The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.     

Noninvasive Assessment of Gene Transfer and Expression by In Vivo Functional and Morphologic Imaging in a Rabbit Tumor Model

Purpose

To evaluate the importance of morphology in quantifying expression after in vivo gene transfer and to compare gene expression after intra-arterial (IA) and intra-tumoral (IT) delivery of adenovirus expressing a SSTR2-based reporter gene in a large animal tumor model.

Materials and Methods

Tumor directed IA or IT delivery of adenovirus containing a human somatostatin receptor type 2A (Ad-CMV-HA-SSTR2A) gene chimera or control adenovirus (Ad-CMV-GFP) was performed in VX2 tumors growing in both rabbit thighs. Three days later, ¹¹¹In-octreotide was administered intravenously after CT imaging using a clinical scanner. ¹¹¹In-octreotide uptake in tumors was evaluated the following day using a clinical gamma-camera. Gene expression was normalized to tumor weight with and without necrosis. This procedure was repeated on nine additional rabbits to investigate longitudinal gene expression both 5 days and 2 weeks after adenovirus delivery. CT images were used to evaluate tumor morphology and excised tissue samples were analyzed to determine ¹¹¹In-octreotide biodistribution ex vivo.

Results

VX2 tumors infected with Ad-CMV-HA-SSTR2 had greater ¹¹¹In-octreotide uptake than with control virus (P<0.05). Intra-arterial and intra-tumoral routes resulted in similar levels of gene expression. Longitudinally, expression appeared to wane at 2 weeks versus 5 days after delivery. Areas of necrosis did not demonstrate significant uptake ex vivo. Morphology identified areas of necrosis on contrast enhanced CT and upon excluding necrosis, in vivo biodistribution analysis resulted in greater percent injected dose per gram (P<0.01) and corresponded better with ex vivo biodistribution(r = 0.72, P<0.01, Coefficient of the x-variable = .72) at 2 weeks than without excluding necrosis (P<0.01).

Conclusion

Tumor specificity and high transgene expression can be achieved in tumors via both tumor directed intra-arterial and intra-tumoral delivery in a large animal tumor model. Using clinical machines, morphologic imaging contributes to functional imaging for quantifying SSTR2-based reporter expression in vivo.     

[18F]-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography of LAPC4-CR Castration-Resistant Prostate Cancer Xenograft Model in Soft Tissue Compartments

Preclinical xenograft models have contributed to advancing our understanding of the molecular basis of prostate cancer and to the development of targeted therapy. However, traditional preclinical in vivo techniques using caliper measurements and survival analysis evaluate the macroscopic tumor behavior, whereas tissue sampling disrupts the microenvironment and cannot be used for longitudinal studies in the same animal. Herein, we present an in vivo study of [¹⁸F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) designed to evaluate the metabolism within the microenvironment of LAPC4-CR, a unique murine model of castration-resistant prostate cancer. Mice bearing LAPC4-CR subcutaneous tumors were administered [¹⁸F]-FDG via intravenous injection. After a 60-minute distribution phase, the mice were imaged on a PET/CT scanner with submillimeter resolution; and the fused PET/CT images were analyzed to evaluate tumor size, location, and metabolism across the cohort of mice. The xenograft tumors showed [¹⁸F]-FDG uptake that was independent of tumor size and was significantly greater than uptake in skeletal muscle and liver in mice (Wilcoxon signed-rank P values of .0002 and .0002, respectively). [¹⁸F]-FDG metabolism of the LAPC4-CR tumors was 2.1 ± 0.8 ID/cm³*wt, with tumor to muscle ratio of 7.4 ± 4.7 and tumor to liver background ratio of 6.7 ± 2.3. Noninvasive molecular imaging techniques such as PET/CT can be used to probe the microenvironment of tumors in vivo. This study showed that [¹⁸F]-FDG-PET/CT could be used to image and assess glucose metabolism of LAPC4-CR xenografts in vivo. Further work can investigate the use of PET/CT to quantify the metabolic response of LAPC4-CR to novel agents and combination therapies using soft tissue and possibly bone compartment xenograft models.     

Imaging of Orthotopic Glioblastoma Xenografts in Mice Using a Clinical CT Scanner: Comparison with Micro-CT and Histology

PurposeThere is an increasing need for small animal in vivo imaging in murine orthotopic glioma models. Because dedicated small animal scanners are not available ubiquitously, the applicability of a clinical CT scanner for visualization and measurement of intracerebrally growing glioma xenografts in living mice was validated.ResultsTumor volumes (mean±SD mm³) were similar between both CT-modalities (micro-CT: 19.8±19.0, clinical CT: 19.8±18.8; Wilcoxon signed-rank test p = 0.813). Moreover, between reader analyses for each modality showed excellent agreement as demonstrated by correlation analysis (Spearman-Rho >0.9; p<0.01 for all correlations). Histologically measured tumor volumes (11.0±11.2) were significantly smaller due to shrinkage artifacts (p<0.05). CNR and SNR were 2.1±1.0 and 1.1±0.04 for micro-CT and 23.1±24.0 and 1.9±0.7 for the clinical CTscanner, respectively.ConclusionClinical CT scanners may reliably be used for in vivo imaging and volumetric analysis of brain tumor growth in mice.     

PET/CT imaging of c-Myc transgenic mice identifies the genotoxic N-nitroso-diethylamine as carcinogen in a short-term cancer bioassay

Background

More than 100,000 chemicals are in use but have not been tested for their safety. To overcome limitations in the cancer bioassay several alternative testing strategies are explored. The inability to monitor non-invasively onset and progression of disease limits, however, the value of current testing strategies. Here, we report the application of in vivo imaging to a c-Myc transgenic mouse model of liver cancer for the development of a short-term cancer bioassay.

Methodology/Principal Findings

μCT and ¹⁸F-FDG μPET were used to detect and quantify tumor lesions after treatment with the genotoxic carcinogen NDEA, the tumor promoting agent BHT or the hepatotoxin paracetamol. Tumor growth was investigated between the ages of 4 to 8.5 months and contrast-enhanced μCT imaging detected liver lesions as well as metastatic spread with high sensitivity and accuracy as confirmed by histopathology. Significant differences in the onset of tumor growth, tumor load and glucose metabolism were observed when the NDEA treatment group was compared with any of the other treatment groups. NDEA treatment of c-Myc transgenic mice significantly accelerated tumor growth and caused metastatic spread of HCC in to lung but this treatment also induced primary lung cancer growth. In contrast, BHT and paracetamol did not promote hepatocarcinogenesis.

Conclusions/Significance

The present study evidences the accuracy of in vivo imaging in defining tumor growth, tumor load, lesion number and metastatic spread. Consequently, the application of in vivo imaging techniques to transgenic animal models may possibly enable short-term cancer bioassays to significantly improve hazard identification and follow-up examinations of different organs by non-invasive methods.     

In Vivo Quantitative Microcomputed Tomographic Analysis of Vasculature and Organs in a Normal and Diseased Mouse Model

Non-bone in vivo micro-CT imaging has many potential applications for preclinical evaluation. Specifically, the in vivo quantification of changes in the vascular network and organ morphology in small animals, associated with the emergence and progression of diseases like bone fracture, inflammation and cancer, would be critical to the development and evaluation of new therapies for the same. However, there are few published papers describing the in vivo vascular imaging in small animals, due to technical challenges, such as low image quality and low vessel contrast in surrounding tissues. These studies have primarily focused on lung, cardiovascular and brain imaging. In vivo vascular imaging of mouse hind limbs has not been reported. We have developed an in vivo CT imaging technique to visualize and quantify vasculature and organ structure in disease models, with the goal of improved quality images. With 1–2 minutes scanning by a high speed in vivo micro-CT scanner (Quantum CT), and injection of a highly efficient contrast agent (Exitron nano 12000), vasculature and organ structure were semi-automatically segmented and quantified via image analysis software (Analyze). Vessels of the head and hind limbs, and organs like the heart, liver, kidneys and spleen were visualized and segmented from density maps. In a mouse model of bone metastasis, neoangiogenesis was observed, and associated changes to vessel morphology were computed, along with associated enlargement of the spleen. The in vivo CT image quality, voxel size down to 20 μm, is sufficient to visualize and quantify mouse vascular morphology. With this technique, in vivo vascular monitoring becomes feasible for the preclinical evaluation of small animal disease models.     

Estimation of carcass composition and cut composition from computed tomography images of live growing pigs of different genotypes

The aim of the present work was (1) to study the relationship between cross-sectional computed tomography (CT) images obtained in live growing pigs of different genotypes and dissection measurements and (2) to estimate carcass composition and cut composition from CT measurements. Sixty gilts from three genotypes (Duroc×(Landrace×Large White), Pietrain×(Landrace×Large White), and Landrace×Large White) were CT scanned and slaughtered at 30 kg (n=15), 70 kg (n=15), 100 kg (n=12) or 120 kg (n=18). Carcasses were cut and the four main cuts were dissected. The distribution of density volumes on the Hounsfield scale (HU) were obtained from CT images and classified into fat (HU between −149 and −1), muscle (HU between 0 and 140) or bone (HU between 141 and 1400). Moreover, physical measurements were obtained on an image of the loin and an image of the ham. Four different regression approaches were studied to predict carcass and cut composition: linear regression, quadratic regression and allometric equations using volumes as predictors, and linear regression using volumes and physical measurements as predictors. Results show that measurements from whole animal taken in vivo with CT allow accurate estimation of carcass and cut composition. The prediction accuracy varied across genotypes, BW and variable to be predicted. In general, linear models, allometric models and linear models, which included also physical measurements at the loin and the ham, produced the lowest prediction errors.     

A potent aminonaphthalimide platinum(IV) complex with effective antitumor activities in vitro and in vivo displaying dual DNA damage effects on tumor cells

A new aminonaphthalimide platinum(IV) complex was developed by incorporating aminonaphthalimide, a DNA intercalator, into the platinum(IV) system. This complex displayed potent antitumor activities against all tested tumor cell lines in vitro and showed great potential in overcoming drug resistance of cisplatin. Moreover, it remarkably inhibited the growth of CT26 xenografts in BALB/c mice without severe side effects in vivo. Then, the compound exhibited a dual DNA damage antitumor mechanism that it could interact with DNA in tetravalent form via the naphthalimide group to cause DNA lesion, and the further liberation of platinum(II) complex after reduction would induce remarkable secondary damage to DNA. Meanwhile, it caused cell apoptosis through an intrinsic apoptosis pathway by up-regulating the expression of caspase 3 and caspase 9.     

In situ activation of tumoricidal properties in rat alveolar macrophages and rejection of experimental lung metastases by intravenous injections of Nocardia rubra cell wall skeleton

Summary The IV injection of squalene-treated cell wall skeleton of Nocardia rubra (N-CWS) into F344 rats rendered their alveolar macrophages (AM) tumoricidal. Maximum tumoricidal activity developed in AM by 24 h after the IV, but not IP or SC, injection of 300 g N-CWS. Tumoricidal activity of AM was maintained for 48–72 h after one IV injection of N-CWS. Experimental lung metastases were produced in female F344 rats by the IV injection of viable syngeneic mammary adenocarcinoma cells. Treatments twice weekly with Hank's balanced salt solution, N-CWS placebo or N-CWS began 3, 7, or 10 days later and were continued for 3 or 4 weeks for a total of six or eight treatments. Practically all the rats (>90%) treated with N-CWS beginning on either day 3 or day 7 after tumor cell challenge survived until day 210, when the experiment was terminated. In contrast, 90% of the rats treated with balanced salt solution or N-CWS placebo died by day 70 of the experiment. Therapy with N-CWS preparation was not successful when the first injection was administered 10 days after tumor cell challenge, suggesting that this therapeutic regimen is effective only against minimal tumor burden. We conclude that in this animal tumor model, the IV injection of N-CWS preparations can render AM tumoricidal and aid in the eradication of pulmonary micrometastases.     

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1^nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.     

Validation of Computed Tomographic Estimates of Intra‐abdominal and Subcutaneous Adipose Tissue in Rats and Mice

We characterized the accuracy, sensitivity, and reliability of computed tomographic (CT) estimates of intra‐abdominal (IA) and subcutaneous (S) adipose tissue (AT) in rats and mice using the Aloka rodent CT. Here, we present the first comparisons of CT estimates of the weights of AT samples ex vivo to balance weights of the same samples, of CT estimates of AT weights in vivo to the weights of resected whole‐body AT, and of CT estimates of the weights of pieces of AT inserted IA or S in vivo to the weights of the same pieces ex vivo. CT underestimated AT weight ex vivo by ～10%, and correction of the automated categorization of IAAT and SAT by Aloka software was required. After these adjustments, correlations (r) of CT estimates and balance weights of resected AT were ≥0.99 in rats and ≥0.92 in mice. CT was impressively sensitive: the 95% probability range of CT estimates of 10,000 mg AT inserts into rats was ±780 mg and of 500 mg inserts into mice, ±20 mg. Scans limited to the abdominal region correlated well (r > 0.90) with whole‐body scan measures of IAAT and SAT in rats and with IAAT, but not SAT (r < 0.80), in mice. Sums of IAAT and SAT correlated well with body weight in rats (r > 0.90), but not in mice (r < 0.80). Coefficients of variance (CVs) of duplicate scans were <5%. We conclude that CT is a valid tool for studies of AT weight in rats and mice, especially when rapid throughput or longitudinal measures are desired.     

In vivo multimodal tumor imaging and photodynamic therapy with novel theranostic agents based on the porphyrazine framework-chelated gadolinium (III) cation

BackgroundA promising strategy for cancer diagnosis and therapy is the development of an agent for multimodal imaging and treatment. In the present paper we report on two novel multifunctional agents prepared on the porphyrazine pigment platform using a gadolinium (III) cation chelated by red-fluorescent tetrapyrrole macrocycles (GdPz1 and GdPz2).MethodsSpectral and magnetic properties of the compounds were analyzed. Monitoring of GdPz1 and GdPz2 accumulation in the murine colon carcinoma CT26 was performed in vivo using fluorescence imaging and MRI. The photobleaching of GdPz1 or GdPz2 and tumor growth rate after photodynamic therapy (PDT) were assessed.ResultsGdPz1 and GdPz2 demonstrated the selective accumulation in tumor that was indicated by higher fluorescence intensity in the tumor area in comparison with the normal tissues. The results of MRI in vivo showed that GdPz1 or GdPz2 provided significant contrast enhancement of the tumor in T1 MR images. PDT with GdPz2 resulted in ~ 20% decrease in fluorescence intensity of the compound and the inhibition of tumor growth.ConclusionsWe assessed the efficiency of two innovative Gd(III) cation-porphyrazine chelates as bimodal MR and fluorescent probes and photosensitizers for PDT and showed their potentials for tumor diagnostics and treatment.General significanceWater-soluble structures simple in preparation and administration into the body represent special interest for theranostics of tumors. Novel porphyrazine macrocycles chelating a central gadolinium cation demonstrated a good prospect as effective multimodal agents, representing a new approach to MRI and fluorescence imaging guided PDT.     

Synthesis and antitumor activity of a new mixed-ligand complex di-n-butyl-(4-chlorobenzohydroxamato)tin(IV) chloride

Reaction of di-n-butyltin(IV) dichloride with 4-chlorobenzohydroxamic acid at 1:1 ratio yielded a new mixed-ligand diorganotin(IV) complex, di-n-butyl-(4-chlorobenzohydroxamato)tin(IV) chloride(DBDCT). It was fully characterized by IR, ¹H, ¹³C, ¹¹⁹Sn NMR spectra and single crystal X-ray analysis. In DBDCT, the tin atom is five-coordinated in a trigonal bipyramidal geometry. DBDCT exhibited strong in vitro cytotoxic activity toward human immature granulocyte leukemia (HL-60), human salivary-gland carcinoma (SGC-7901), human henrietta carcinoma (Hela) and human urinary bladder (T24) cell lines which, in some cases, were equal to, or even higher than those of cis-dichlorodiammineplatinum(II) (cisplatin, DDP), the widely clinically used drug. The further in vivo antitumor tests of DBDCT towards the transplantation tumor models of sarcoma carcinoma (S₁₈₀), hepatocellular carcinoma (H₂₂) and Ehrlich’s ascites carcinoma (EAC) on mice were carried out via injection intraperitoneally with cisplatin as positive contrast drug. The results showed that DBDCT displayed in vivo antitumor activity against the hepatocellular carcinoma H₂₂ and sarcoma carcinoma S₁₈₀ which were close to those of cisplatin, meanwhile, the survival-extending rates at middle dose and high dose on mice Ehrlich’s ascites tumor EAC were higher than those of cisplatin, and there was a good dose-effect relationship.     

Over-Expression of miR-106b Promotes Cell Migration and Metastasis in Hepatocellular Carcinoma by Activating Epithelial-Mesenchymal Transition Process

Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.     

Peptide-Based Optical uPAR Imaging for Surgery: In Vivo Testing of ICG-Glu-Glu-AE105

Near infrared intra-operative optical imaging is an emerging technique with clear implications for improved cancer surgery by enabling a more distinct delineation of the tumor margins during resection. This modality has the potential to increase the number of patients having a curative radical tumor resection. In the present study, a new uPAR-targeted fluorescent probe was developed and the in vivo applicability was evaluated in a human xenograft mouse model. Most human carcinomas express high level of uPAR in the tumor-stromal interface of invasive lesions and uPAR is therefore considered an ideal target for intra-operative imaging. Conjugation of the flourophor indocyanine green (ICG) to the uPAR agonist (AE105) provides an optical imaging ligand with sufficiently high receptor affinity to allow for a specific receptor targeting in vivo. For in vivo testing, human glioblastoma xenograft mice were subjected to optical imaging after i.v. injection of ICG-AE105, which provided an optimal contrast in the time window 6–24 h post injection. Specificity of the uPAR-targeting probe ICG-AE105 was demonstrated in vivo by 1) no uptake of unconjugated ICG after 15 hours, 2) inhibition of ICG-AE105 tumor uptake by a bolus injection of the natural uPAR ligand pro-uPA, and finally 3) the histological colocalization of ICG-AE105 fluorescence and immunohistochemical detected human uPAR on resected tumor slides. Taken together, our data supports the potential use of this probe for intra-operative optical guidance in cancer surgery to ensure complete removal of tumors while preserving adjacent, healthy tissue.     

Inhibitory effect of caffeic acid phenethyl ester on mice bearing tumor involving angiostatic and apoptotic activities

This study aims at investigating the anti-tumor effect of caffeic acid phenethyl ester (CAPE) against animal carcinogenesis. In order to substantiate this fact implanted tumor Ehrlich carcinoma cells were assessed in vivo to Swiss mice strain. We found that administrating of CAPE (15 mg/kg S.C.) showed that the tumor volume decreased significantly by 51%. As a result, it improved animal chances of survival and they became healthier. An anti-angiogenic effect of CAPE in vivo was observed, as determined by a significant serum matrix metalloproteinase (MMP-9) reduction (142.1 ng/ml), activation of endostatin serum level (1.9 ng/ml), as well as DNA fragmentation in tumor treated mice when compared with untreated ones. Conclusion: CAPE has a significant inhibitory effect on tumor in vivo. This inhibition may be related to its angiostatic and apoptotic effects. It also reduced angiogenic factors which may shift the equilibrium to the angiostatic effect of CAPE. These findings provide the possibility for the future use of CAPE as tumor therapy in human clinical trials.     

Embelin (2,5-dihydroxy-3-undecyl-p-benzoquinone): A bioactive molecule isolated from Embelia ribes as an effective photodynamic therapeutic candidate against tumor in vivo

The present study was carried out to assess the photosensitizing potential of embelin, the biologically active natural product isolated from Embelia ribes in photodynamic therapy (PDT) experiments in vivo. In vitro PDT clearly indicated that embelin recorded significant cytotoxicity in Ehrlich's Ascites Carcinoma (EAC) cells, which is superior to 5-aminolevulinic acid, a known photodynamic compound. For in vivo experiments solid tumor was induced using EAC cells in the male Swiss albino mice of groups I, II, III and IV. Group I served as the control (without solid tumor), group II served as tumor bearing mice without treatment and groups III and IV served as treatments. At the completion of 4 weeks of induction, the tumor bearing mice from group III and IV were given an intraperitoneal injection with embelin (12.5 mg/kg body weight). After 24 h, tumor area in the Group III and IV animals was exposed to visible light from a 1000 W halogen lamp. The mice from groups I to III were sacrificed 2 weeks after the PDT treatment and the marker enzymes (myeloperoxidase [MPO], β-d-glucuronidase, and rhodanese) were assayed and expression of Bcl-2 and Bax were analyzed in normal and tumor tissues. Animals from group IV were sacrificed after 90 days of PDT treatment and the above mentioned parameters were recorded. Reduction in tumor volume and reversal of biochemical markers to near normal levels were observed in the treated groups. This is the first report on PDT using a natural compound for solid tumor control in vivo. The uniqueness of the mode of treatment lies in the selective uptake of the nontoxic natural compound, embelin from the medicinal plant E. ribes used in Indian system of medicine, by the solid tumor cells and their selective destruction using PDT without affecting the neighboring normal cells, which is much advantageous over radiation therapy now frequently used.     

The Cardioprotective Protein Apolipoprotein A1 Promotes Potent Anti-tumorigenic Effects

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b⁺ F4/80⁺ macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.     

Non-Invasive Imaging of Tumors by Monitoring Autotaxin Activity Using an Enzyme-Activated Near-Infrared Fluorogenic Substrate

Autotaxin (ATX), an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2) that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.