Histological Sections Of Tumors Research Articles

Chemokine CXCL8 and its receptors as markers of colorectal cancer

BACKGROUND: The chemokine CXCL8 and its receptor are involved in the activation and transport of inflammatory mediators and regulate the proliferation and renewal of cancer stem cells in colon cancer. It is believed that CXC signaling may be associated with poor prognosis in colorectal cancer. AIM: To study the possibilities of using CXCL8-CXCR1/2 pathway indicators as markers in colorectal cancer. MATERIAL AND METHODS: Isolation of ribonucleic acid (RNA) from histological sections of tumors obtained intraoperatively from 59 patients diagnosed with colorectal cancer was carried out using magnetic particles, and quantitative polymerase chain reaction in real time was performed. Calculation of the normalized expression of the CXCL8 and CXCR1 genes relative to the referee gene was done using special software. Statistical data processing was performed using Statistica 3.0, BioStat v. 7.1 programs. Comparisons of characteristics were performed using the Mann–Whitney U test. Cox and Kaplan–Meier criteria were used to analyze overall and disease-free survival. RESULTS: The expression of CXCL8 in intestinal adenocarcinoma cells with low differentiation [Me (Q1–Q3) — 8.770 (1.127–1.114)] was significantly higher than in the groups of moderately and well differentiated tumors (p=0.004 and p=0.012, respectively); and in tumor tissue refractory to chemotherapy, it was significantly higher [4.374 (2.052–7.045)] compared to resistant [2.200 (1.388–5.037); p=0.008] and sensitive [1.624 (0.739–2.586); p=0.042]. The level of CXCR1 messenger RNA in tumor tissue was increased in the presence of BRAF mutation [3.645 (0.801–1.090); p=0.009] and low tumor differentiation [6.965 (3.938–12.225); p=0.002], as well as in tumor tissue refractory to FOLFOX/XELOX chemotherapy [46.224 (27.580–83.570); p=0.0009]. CONCLUSION: Expression of components of the CXCL8-CXCR1/2 signaling pathway in tumor tissue may be a marker of sensitivity to FOLFOX/XELOX chemotherapy in colorectal cancer.

Effect of FAPα-expressing tumor cell vaccine with PD-1 mAb on its efficacy in mice colorectal cancer model.

e14615 Background: Colorectal cancer (CRC) is the third prevalent malignant tumor all over the world, and many cases are metastatic at diagnosis. Immunotherapy emerges a promising treatment in CRC. However, anti-PD-1 monotherapy alone has not yielded a clinical benefit in majority of these patients. Therefore, combination therapy is necessary to overcome this limitation. Previously, we have developed a FAPα-expressing tumor vaccine and demonstrated its anti-tumor effects by reversing of immunosuppressive tumor microenvironment (TME). In the current study, we hypothesized that combination therapy could enhance the effect of anti-PD1 therapy. Methods: FAPα-expressing CT26 cells were produced as previously described. To establish xenograft tumor model, CT26 cells were injected into left flank of BALB/c mice. Anti-PD-1 monoclonal antibodies(mAb) were injected intraperitoneally twice weekly up to 5 injections. FAPα-vaccines were injected subcutaneously around tumors weekly up to 3 injections. Histological sections of tumours were analysed by H&E staining and immunofluorescence. Flow cytometric analysis was done using an FACSCalibur (BD) and analyzed using FlowJo software (TreeStar). Gene expression profile in TME was analyzed by bulk RNA sequencing. Results: In mice xenograft CT26 tumor model, either anti-PD-1 immunotherapy or FAPα tumor cell vaccine produced suppression of CT26 tumor growth compared with the control group. However, combining anti-PD-1 mAb and FAPα-expressing tumor cell vaccine elicited the most remarkable anti-tumor effect and induced the most pronounced tumor growth suppression among all groups. In combination therapy group of the vaccine and PD-1 mAb, there were significantly more infiltrated lymphocytes and less FAPα+ fibroblasts. Flow cytometry demonstrated that the combination therapy promoted M1 macrophage polarization with a reduction of M2-like tumor associated macrophages (TAMs) and an increase of M1-like TAMs. This polarization was in consistent with the reduced level of IL-10 that is predominant cytokine secreted by M2 macrophages. A reduction of myeloid-derived suppressor cells (MDSCs) in the TME were also observed. Using bulk RNAseq we observed a set of immune related genes upregulated following combination therapy, including innate immune response, antigen presentation, lymphatic cells and recruitment and T cells activation. Decreased secretion of immune inhibitory chemokines (eg, CXCL12, IL-10, TGFβ) was also found by ELISA in tumors from mice treated with combination therapy. Conclusions: Our preliminary results suggested that combination therapy of FAPα-expressing tumor vaccine and anti-PD-1 therapy is a promising way to improve the efficacy of immunotherapy through modulation of immunosuppressive TME. In the future, more research is needed to further elucidate mechanism and clinical value of this combination approach.

PET/MRI of hypoxia and vascular function in ER-positive breast cancer: correlations with immunohistochemistry

ObjectivesTo explore the relationship between indices of hypoxia and vascular function from 18F-fluoromisonidazole ([18F]-FMISO)-PET/MRI with immunohistochemical markers of hypoxia and vascularity in oestrogen receptor–positive (ER +) breast cancer.MethodsWomen aged > 18 years with biopsy-confirmed, treatment-naïve primary ER + breast cancer underwent [18F]-FMISO-PET/MRI prior to surgery. Parameters of vascular function were derived from DCE-MRI using the extended Tofts model, whilst hypoxia was assessed using the [18F]-FMISO influx rate constant, Ki. Histological tumour sections were stained with CD31, hypoxia-inducible factor (HIF)-1α, and carbonic anhydrase IX (CAIX). The number of tumour microvessels, median vessel diameter, and microvessel density (MVD) were obtained from CD31 immunohistochemistry. HIF-1α and CAIX expression were assessed using histoscores obtained by multiplying the percentage of positive cells stained by the staining intensity. Regression analysis was used to study associations between imaging and immunohistochemistry variables.ResultsOf the lesions examined, 14/22 (64%) were ductal cancers, grade 2 or 3 (19/22; 86%), with 17/22 (77%) HER2-negative. [18F]-FMISO Ki associated negatively with vessel diameter (p = 0.03), MVD (p = 0.02), and CAIX expression (p = 0.002), whilst no significant relationships were found between DCE-MRI pharmacokinetic parameters and immunohistochemical variables. HIF-1α did not significantly associate with any PET/MR imaging indices.ConclusionHypoxia measured by [18F]-FMISO-PET was associated with increased CAIX expression, low MVD, and smaller vessel diameters in ER + breast cancer, further corroborating the link between inadequate vascularity and hypoxia in ER + breast cancer.Key Points• Hypoxia, measured by [18F]-FMISO-PET, was associated with low microvessel density and small vessel diameters, corroborating the link between inadequate vascularity and hypoxia in ER + breast cancer.• Increased CAIX expression was associated with higher levels of hypoxia measured by [18F]-FMISO-PET.• Morphologic and functional abnormalities of the tumour microvasculature are the major determinants of hypoxia in cancers and support the previously reported perfusion-driven character of hypoxia in breast carcinomas.

S-100 PROTEIN EXPRESSION LEVELS AS A POTENTIAL PROGNOSTIC MARKER IN NEUROBLASTOMA

Objective: S-100 is an acidic-calcium-binding protein and was first described in cells of neuroendocrine origin. It plays an important role in various cellular processes such as cell differentiation and proliferation. S-100 is described to be a negative prognostic marker for the advanced stages of melanoma and was evaluated to be prognostic biomarker for different cancers. Neuroblastoma(NB) is remarkable for its broad spectrum of clinical behavior, varying from spontaneous regression to progressive disease and accounts for ~15% of childhood cancer‐related mortality. This diversity in behavior correlates closely with defined clinical and biologic features and combinations of prognostic variables are used for risk-group assignment. The aim of this study is to investigate the S-100 expression levels among neuroblastoma patients and to determine if S-100 expression levels could be prognostic biomarker for neuroblastoma. Materials and Methods: Histological tumor sections of 40 cases of varying stages and risk groups of neuroblastoma were studied. The patients were staged according to criteria defined by Turkish Pediatric Oncology Group and International Neuroblastoma Staging System(INSS). Risk groups were determined according to the International Neuroblastoma Risk Group(INRG) classification system. S-100 protein expression levels were evaluated by immunohistochemical staining on paraffin sections of neuroblastoma patient tissue samples. All sections were independently and blindly studied for protein expression under microscopic examination. P values<0.05 was accepted statistically significant. Results: Schwann cells in the ganglioneuromas and ganglioneuroblastomas always strongly stained. Neuroblastoma cases with early stage and low-to-intermediate risk showed upregulated expression. Conclusion: While study is still ongoing, preliminary results showed that S-100 could be a candidate prognostic marker for neuroblastoma patients.

INVESTIGATION OF THE POTENTIAL EFFECTS OF YAP-1 AND NESTIN ON RISK CLASSES AND PROGNOSIS IN NEUROBLASTOMA

Introduction: Neuroblastoma (NB) is the most common solid tumors in childhood. MYCN is the main prognostic factor for risk classification and staging in NB patients. YAP (Yes Associated Protein) plays a critical role in many types of cancer. In NB, YAP expression levels may be correlate with advanced tumor staging. Nestin is an important neuronal differentiation and cancer stem cell marker in NB. The aim of this study is to examine the potential roles of YAP-1 and Nestin expressions in terms of prognosis and risk classifications for neuroblastoma. Materials and Methods: Histological tumor sections of 55 cases were examined. YAP-1 and Nestin protein expression levels were evaluated by immunohistochemical staining on paraffin sections of different neuroblastoma risk groups. YAP-1 and Nestin expression levels were evaluated with clinical findings of NB patients. P value was <0.05 accepted as a statistically significant. Results: 55 patient tissues were included in this study, of which 24 were male and 21 were female. The ages of the patients are between 1 month to 11 years. YAP-1 protein was expressed in 36.4% in MYCN(+)and 14.6% in MYCN(-) patients. Nestin protein was expressed 36.4% in MYCN(+), and 20.6% in MYCN(-) patients. YAP-1 was expressed 18% in all tissues. Nestin expressions were determined 20.4% in all tissues. YAP-1 was expressed in 26.9% in high-risk and 11.5% in low-risk groups. Nestin was expressed 24.4% in high-risk and 33.3% in low-risk groups. Conclusion: YAP-1 and Nestin might be a clinically risk markers for neuroblastoma patients.

Association Between GATA3 and Histopathological and Immunohistochemical Parameters in Early-Infiltrating Breast Carcinomas

This study evaluated the frequency of GATA-binding protein 3 (GATA3) expression in early breast cancer and its relationship with histopathological and immunohistochemical parameters. GATA3 was analysed by immunohistochemistry in histological sections of tumors from 105 female patients, with histological diagnosis of invasive breast carcinoma (BC), at clinical stages I, II and IIIA, who underwent primary surgical treatment. GATA3 nuclear expression was determined as the percentage of positive tumor cells and further categorized as high (positive expression in more than 95% of cells) or non-high (negative or low positive expression in up to 95% of tumor cells). GATA3 expression was analysed according to the patient age, tumor and node pathological stage, histological type, histological and nuclear grade, lymphovascular invasion, and estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), human epidermal growth factor 2 (HER2) status, and Ki-67 expression. GATA3 expression was positive in 103 cases (98.1%). High expression was significantly associated with low histological and nuclear grade, positive hormonal receptors, and less proliferative activity based on Ki-67 expression. A prominent feature was that 94.7% of the ER-positive/HER2-negative cases presented high-GATA3 expression, as 94.0% of the tumors showing high-GATA3 were ER-positive. In ER-negative/HER2-positive or ER-negative/HER2- negative, high-GATA3 was present in 25% while 75% were non-high-GATA3 compared with ER-positive/HER2- negative (4.1%) and ER-positive/HER2-positive (20%). Proliferative activity in triple-negative breast cancer tended to be higher among tumors with low-GATA3, irrespective of AR expression. In the group of ER-positive/HER2-negative tumors only three cases were low-GATA3 (85% and 80%), both with high proliferative activity. High GATA3 expression is associated with favorable histopathologic and immunohistochemical BC prognostic factors.

Vasculogenic mimicry correlates to presenting symptoms and mortality in uveal melanoma

PurposeFluid-conducting extracellular matrix patterns known as vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma and other cancers. We investigate the correlations between VM, presenting symptoms, mortality, and the area density of periodic acid-Schiff positive histological patterns (PAS density).MethodsSixty-nine patients that underwent enucleation for uveal melanoma between 2000 and 2007 were included. Clinicopathological parameters presenting symptoms and outcomes were collected. Histological tumor sections were evaluated for VM and PAS density was quantified with digital image analysis.ResultsThirty-four patients (49%) presented with blurred vision. 18 (26%) with a shadow in the visual field, 7 (10%) with photopsia and/or floaters, and 2 (3%) with metamorphopsia. Nine patients (13%) had no symptoms at all. Median follow-up was 16.7 years (SD 2.6). A shadow in the visual field, but no other symptom, was positively correlated with the presence of VM (φ 0.70, p < 0.001) and greater PAS density (p < 0.001). In multivariate regression, retinal detachment (RD), presence of VM, and PAS density ≥ median were independent predictors of a shadow, but not tumor distance to the macula, tumor apical thickness, tumor diameter, or ciliary body engagement. The presence of VM was associated with significantly shorter cumulative disease-specific survival (Wilcoxon p = 0.04), but not PAS density ≥ median, presenting symptoms or RD (p > 0.28).ConclusionTumors from uveal melanoma patients that report a visual field shadow are likely to display VM and greater PAS density, likely explaining the previously reported association between this symptom and poor prognosis.

Contrast-enhanced ultrasound for evaluation of tumor perfusion and outcome following treatment in a murine melanoma model

Due to a lack of data on predictors of electroporation-based treatment outcomes, we investigated the potential predictive role of contrast-enhanced harmonic ultrasound (CEUS) in mice B16F10 melanoma treated by gene electrotransfer (GET) to silence melanoma cell adhesion molecule (MCAM) and radiotherapy, which has not been evaluated yet. CEUS evaluation was verified by tumor histological analysis. Mice bearing subcutaneous tumors were treated with GET to silence MCAM, irradiation or the combination of GET to silence MCAM and irradiation (combined treatment). CEUS of the tumors used to evaluate tumor perfusion was performed before and up to 10 days after the beginning of the experiment, and the CEUS results were compared with tumor growth and the number of blood vessels analyzed in the histological tumor sections. CEUS revealed a decrease in tumor perfusion in the combined therapy groups compared with the control groups and correlated with tumor histological analyses, which showed a decreased vascular density. In this study a trend of inverse correlation was observed between tumor perfusion and treatment efficacy. The greater the perfusion of the tumor, the shorter the expected doubling time. Furthermore, decreased perfusion showed a trend to correlate with higher antitumor efficacy. Thus, CEUS could be used to predict tumoral vascular density and treatment effectiveness.

The Phytosterol Peniocerol Inhibits Cell Proliferation and Tumor Growth in a Colon Cancer Xenograft Model.

Objective: This study aimed to evaluate the cytotoxic activity of peniocerol against human colon cancer cell lines and its antitumor effect in vivo in a xenograft model using nu/nu mice.Materials and Methods: SW-620, HCT-15, and HCT-116 colon cancer cell lines were treated with peniocerol for cytotoxicity by crystal violet technique. Cell apoptosis induction was detected by flow cytometry, and the antitumor activity of peniocerol was evaluated in a xenograft model of HCT-116 in nu/nu mice. After treatment, the effect of peniocerol was analyzed in histological sections of tumors by immunohistochemistry using DAPI, anti-PCNA, and PARP-1 antibodies.Results: Peniocerol inhibited cell growth and induced apoptosis in vitro in a time and dose-dependent manner. Besides, peniocerol administration (30 or 15 mg/kg) inhibited tumor growth and induced apoptosis in the xenograft mice. The lack of peniocerol toxicity was proved by a biochemical blood analysis of healthy nu/nu mice administrated with this sterol.Conclusions: Our results proved that peniocerol induces apoptosis in vitro and in vivo assays.

Prognostic significance of lymphovascular invasion in patients with esophageal squamous cell carcinoma treated with neoadjuvant chemoradiotherapy.

To investigate the prognostic significance of lymphovascular invasion (LVI) in patients with esophageal squamous cell carcinoma (ESCC) treated with neoadjuvant chemoradiotherapy (nCRT). We retrospectively reviewed the charts of 416 consecutive patients with ESCC who underwent surgical resection following nCRT at the Chang Gung Memorial Hospital between 1998 and 2008. After exclusion of patients with non-R0 resection or showing no residual tumor (ypT0Nx), the histological tumor sections of 231 patients were reviewed for LVI. Univariate and multivariate analyses were used to identify the independent predictors of overall survival (OS). LVI was observed in 85 patients (36.8 %). The presence of LVI was related to close circumferential resection margins (CRMs), a greater depth of invasion, poor tumor differentiation, and an increased occurrence of lymph node metastases (LNM). The 5 year OS was significantly lower (10 %) in patients with LVI than in those without (31 %; p < 0.001). By multivariate Cox regression analyses, LVI (hazard ratio [HR] 1.5; p = 0.002) and LNM (HR 1.6; p = 0.007) were identified as independent adverse prognostic factors for OS. The 5 year OS rates according to the number of risk factors present were 35, 21, 20, and 5 for LVI(-)LNM(-), LVI(+)LNM(-), LVI(-)LNM(+), and LVI(+)LNM(+) patients, respectively (p < 0.001). The presence of LVI is independently associated with shorter OS in ESCC patients receiving nCRT. Links between LVI and LNM may provide new clues for the prognostic stratification of esophageal cancer.

An automatic algorithm for the segmentation and morphological analysis of microvessels in immunostained histological tumour sections

A fully automatic segmentation and morphological analysis algorithm for the analysis of microvessels from CD31 immunostained histological tumour sections is presented. Development of the algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, to provide the seeds for a 'region-growing' method for object segmentation in the 3D hue, saturation, value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing objects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels. The automatic segmentation was validated against a hand-segmented set of 44 images from three different SW1222 human colorectal carcinomas xenografted into mice. 96.3 ± 0.9% of pixels were found to be correctly classified. Automated segmentation was carried out on a further 53 images from three histologically distinct mouse fibrosarcomas (MFs) for morphological comparison with the SW1222 tumours. Four morphometric measurements were calculated for each segmented vessel: vascular area (VA), ratio of lumen area to vascular area (lu/VA), eccentricity (e), and roundness (ro). In addition, the total vascular area relative to tumour tissue area (rVA) was calculated. lu/VA, e and ro were found to be significantly smaller in MF tumours than in SW1222 tumours (p < 0.05; unpaired t-test). The algorithm is available through the website http://www.caiman.org.uk where images can be uploaded, processed and sent back to users. The output from CAIMAN consists of the original image with boundaries of segmented vessels overlaid, the calculated parameters and a Matlab file, which contains the segmentation that the user can use to derive further results.

Tumor Regression and Curability of Preclinical Neuroblastoma Models by PEGylated SN38 (EZN-2208), a Novel Topoisomerase I Inhibitor

Treatment of neuroblastoma is successful in less than half of patients with high-risk disease. The antitumor activity of a water soluble pegylated SN38 drug conjugate, EZN-2208, was compared with CPT-11 (a prodrug for SN38) in preclinical models of human neuroblastoma. The in vitro cytotoxicity of EZN-2208 was tested by counting trypan blue dye- and Annexin V-positive cells, whereas its therapeutic efficacy was evaluated, in terms of survival, and antitumor and antiangiogenic activities, in s.c. luciferase-transfected, pseudometastatic, and orthotopic neuroblastoma animal models. EZN-2208 was about 100-fold more potent than CPT-11 in vitro, by inducing apoptosis/necrosis and p53 expression and by reducing hypoxia-inducible factor (HIF)-1α/HIF-2α expression. EZN-2208 gave superior antitumor effects compared with CPT-11 in neuroblastoma xenografts. EZN-2208 treatment always resulted in lack of tumor detection at the end of trials whereas only small therapeutic effects were observed with CPT-11, as assessed by luciferase assay or tumor size, or even by staining histologic sections of tumors with antibodies recognizing neuroblastoma cells and cell proliferation. In a neuroblastoma model resistant to doxorubicin, cisplatin, vincristine, fenretinide, and topotecan, EZN-2208 induced 100% curability. It also blocked tumor relapse after topotecan-vincristine-doxorubicin combined treatment. Mechanistic experiments showed statistically significantly enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Histone H2ax staining as well as decreased vascular endothelial growth factor, CD31, matrix metalloproteinase (MMP)-2, and MMP-9 expression in tumors removed from EZN-2208-treated mice and radiating vessels invading the tumor implanted onto the chorioallantoic membranes. EZN-2208 should be considered a most promising novel antineuroblastoma agent. An ongoing phase I study in pediatric patients should identify the optimal dose for a phase II study.

Iron oxide core oil-in-water emulsions as a multifunctional nanoparticle platform for tumor targeting and imaging

Nanoemulsions are increasingly investigated for the delivery of hydrophobic drugs to improve their bioavailability or make their administration possible. In the current study, oil-in-water emulsions with three different mean diameters (30, 60, and 95 nm) were developed as a new multimodality nanoparticle platform for tumor targeting and imaging. To that aim, hydrophobically coated iron oxide particles were included in the soybean oil core of the nanoemulsions to enable their detection with magnetic resonance imaging (MRI), while the conjugation of a near infrared fluorophore allowed optical imaging. The accumulation of this novel nanocomposite in subcutaneous human tumors in nude mice was demonstrated with MRI and fluorescence imaging in vivo, and with Perl's staining of histological tumor sections ex vivo.

Whole Genome Amplification for Array Comparative Genomic Hybridization Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Histological Sections

Array comparative genomic hybridization (CGH) is useful to assess genome-wide chromosomal imbalance, but the requirement for relatively large amounts of DNA can be a limitation, in particular for samples extracted from small tumor areas on paraffin sections. Whole genome amplification (WGA) can be performed before array CGH to obtain sufficient DNA, but the possibility of artifacts attributable to biased amplification cannot be excluded. We optimized the WGA protocol to generate sufficient DNA with minimum amplification bias. Using formalin-fixed, paraffin-embedded histological sections of tumors carrying known TP53 mutations, LOH 1p, LOH 10q, LOH 19q, and EGFR amplification, we first optimized the protocol so that these genetic alterations were detected after WGA. We found that a ligation step before WGA is important because it allows a short reaction time with Phi29 to generate WGA-DNA with greatly decreased amplification bias. Using template >150 ng of DNA, a ligation step before WGA, and a short reaction time with Phi29 DNA polymerase (<1.5 hours), we obtained WGA-DNA (>4 mug) with minimum amplification bias (less than threefold). Using this protocol, we performed array CGH (Agilent 105K) before and after WGA. Pearson correlation analysis indicated a significant positive correlation in array CGH results between DNA before and after WGA (P < 0.0001). These results suggest that genetic analyses are possible using WGA-DNA extracted from paraffin sections, but that they should be performed with a carefully optimized and controlled protocol.

Plasminogen fragment K1–3 inhibits expression of adhesion molecules and experimental HCC recurrence in the liver

There is no established adjuvant or neo-adjuvant treatment to curb tumor recurrence of hepatocellular carcinoma (HCC). Recent data showed that angiostatic factors can inhibit tumor cell adhesion to the endothelium and therefore recurrence/metastasis. We tested a potential preventive, pre-operative strategy using plasminogen kringles 1-3 (K1-3) to overcome this hurdle. Effects of K1-3 on the intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) expression was analyzed in vitro and in vivo on RNA and protein levels. Influence of K1-3 on HCC recurrence in the liver was analyzed in an orthotopic tumor model. K1-3 decreased ICAM expression in Hepa129 tumor cells and VCAM expression in SVEC4-10 endothelial cells in vitro. In vivo, ICAM was reduced in histological tumor sections. Preventive treatment with AdK1-3 inhibited experimental HCC recurrence and tumor growth in the liver. We were able to show that K1-3 inhibits intrahepatic tumor recurrence. This novel aspect elucidates a possible approach to prevent HCC recurrence.

Treatment of solid tumors by interstitial release of recoiling short-lived alpha emitters

A new method utilizing alpha particles to treat solid tumors is presented. Tumors are treated with interstitial radioactive sources which continually release short-lived alpha emitting atoms from their surface. The atoms disperse inside the tumor, delivering a high dose through their alpha decays. We implement this scheme using thin wire sources impregnated with 224Ra, which release by recoil 220Rn, 216Po and 212Pb atoms. This work aims to demonstrate the feasibility of our method by measuring the activity patterns of the released radionuclides in experimental tumors. Sources carrying 224Ra activities in the range 10–130 kBq were used in experiments on murine squamous cell carcinoma tumors. These included gamma spectroscopy of the dissected tumors and major organs, Fuji-plate autoradiography of histological tumor sections and tissue damage detection by Hematoxylin-Eosin staining. The measurements focused on 212Pb and 212Bi. The 220Rn/216Po distribution was treated theoretically using a simple diffusion model. A simplified scheme was used to convert measured 212Pb activities to absorbed dose estimates. Both physical and histological measurements confirmed the formation of a 5–7 mm diameter necrotic region receiving a therapeutic alpha-particle dose around the source. The necrotic regions shape closely corresponded to the measured activity patterns. 212Pb was found to leave the tumor through the blood at a rate which decreased with tumor mass. Our results suggest that the proposed method, termed DART (diffusing alpha-emitters radiation therapy), may potentially be useful for the treatment of human patients.

A New Preclinical In-Vivo System for Studying and Testing Chemotherapy on Human Hematological Malignancies.

Transplantation of cells and tissues to the embryonic chick, especially to its chorioallantoic membrane (CAM), has been used for many years to study the vascularization and growth of solid mammalian tumors. However, it has not previously been used to study mammalian hematological malignancies. We have now examined the potential of the pre-immunocompetent chick embryo as a host for human leukemias. Two human myeloid leukemia lines, K562 and DAMI, and the T-cell ALL line Jurkatt were engineered to stably express eGFP. K562 erythroleukemia cells introduced either by injection into the yolk sac or by layering onto the CAM, showed inconsistent engraftment. By contrast, K562 and DAMI cells engrafted in 100% of embryos following intravascular (iv) or intra-amniotic injection. The engraftment of these human leukemia cell lines following iv injection was rapid, appearing as soon as 7 days after injection into the embryos. This novel methodology is quite rapid compared to the standard xenografting model of SCID mice, which requires 6–8 weeks for engraftment of human leukemia after intravenous injection. Engraftment was visualized in-vivo as whitish tumor nodules in the avian chorioallantoic membrane (CAM). eGFP expression enhanced the detection of the tumors in terms of both speed and sensitivity. Leukemia cells were consistently detected by PCR amplification of human-specific α-satellite DNA sequences in the hematopoietic organs: bone marrow (BM), liver and spleen, as well as in the brain. DAMI cells also consistently engrafted in the embryos' hematopoietic organs, but at lower levels than K562. In contrast, Jurkatt cells did not show consistent engraftment. K562 cells were immunopositive for human CD45 and human mitochondrial and lysosomal antigens in histological sections of tumors in the CAM. K562 cells were also detected by immunostaining of peripheral blood smears from engrafted embryos. In one case, human cells were detected by PCR in the BM of the embryos one week after injection of a patient sample of fresh lymphoma cells. Engraftment levels of iv-injected K562 and DAMI were increased more than 100-fold (real-time qPCR) by using avian eggs with longer incubation periods than the chick to extend the graft period to 14 days. The potential use of this system for testing cytotoxic therapy was also investigated. Dramatic and consistent regression of tumors in the CAM was induced by a single dose of doxorubicin administered to K562-engrafted embryos. These results demonstrate for the first time that the chick embryo can be used for study of human hematological malignancies. This new platform has the advantages of significantly lower cost and the lack of requirement of sentient animals when compared to the other in-vivo models of transplantation to SCID mice and large mammalian embryos. In addition, grafting human blood malignancies into the chick embryo has the potential to provide a rapid assay for testing chemotherapies, which could significantly reduce drug development time and expense.

Decreased B16F10 melanoma growth and impaired tumour vascularization in BDF1 mice with quercetin-cyclodextrin binary system

The aim of this work was to study the inclusion behaviour of a poorly water-soluble bioflavonoid, quercetin, towards sulfobutyl ether-7beta-cyclodextrin. It also involves angiogenesis inhibition in-vivo in addition to in-vitro human cancer cell growth inhibition study of quercetin and its cyclodextrin complex. Drug-cyclodextrin solid inclusion complexes were prepared and characterized in solution and in the solid state. An in-vitro anti-proliferation study using plain drug and its solubilized form was carried out on human cancer cell lines of different origin. Further, an in-vivo tumour growth inhibition study was carried out using a mouse melanoma model. Histological sections of tumours were examined for the evaluation of tumour microvessel density. Significant enhancement of the solubility and dissolution rate of the quercetin, which occurred after complexation, might be attributed to the decrease in crystallinity of drug. SBE7betaCD complex of quercetin was more potent for inhibiting cell proliferation in human erythroleukaemia and cervix cancer cells. Decreased tumour microvessel density in mouse melanoma after oral quercetin administration led to diminished tumour cell proliferation. Quercetin-SBE7betaCD complex showed significantly improved anti-cancer activity at much lower concentration than the plain drug, providing evidence for dose reduction without affecting therapeutic efficacy when using cyclodextrin carriers.

Dynamic contrast-enhanced magnetic resonance imaging is a poor measure of rectal cancer angiogenesis

The aim of this study was to investigate the use of magnetic resonance imaging (MRI) for non-invasive measurement of rectal cancer angiogenesis and hypoxia. Fifteen patients with rectal adenocarcinoma underwent preoperative dynamic contrast-enhanced (DCE) and blood oxygenation level-dependent (BOLD) MRI. Microvessel density (CD31 level), and expression of vascular endothelial growth factor (VEGF) and carbonic anhydrase (CA) 9 were measured immunohistochemically in histological tumour sections from 12 patients. Serum VEGF levels were also measured in 14 patients. Correlations between quantitative imaging indices and immunohistochemical variables were examined. There was good correlation between circulating VEGF and CD31 expression (r(S) = 0.88, P < 0.001). CD31 expression did not correlate with any dynamic MRI parameter, except transfer constant, with which it correlated inversely (r(S) = -0.65, P = 0.022). Tissue and circulating VEGF levels did not correlate, and neither correlated with any tumour DCE MRI parameter. No relationship was seen between BOLD MRI and CA-9 expression. The negative correlation between transfer constant (reflecting tumour blood flow and microvessel permeability) with CD31 expression is paradoxical. DCE MRI methods for assessing tissue vascularity correlate poorly with histological markers of angiogenesis and hypoxia, suggesting that DCE MRI does not simply reflect static histological vascular properties in patients with rectal cancer.

Quantitative Analysis of Chemotherapeutic Effects in Tumors Using In Vivo Staining and Correlative Histology

Aims: To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis. Methods: Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution. Results: We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be ∼2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a ∼1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line. Conclusions: Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.