Disposition characteristics of mitomycin C-dextran conjugate in normal and tumor-bearing muscles of rabbits

Disposition characteristics of the macromolecular prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), in normal and tumor (VX2 carcinoma)-bearing rabbit thigh muscles were studied using the in situ vascular perfusion technique. Three types of cationic MMC-D (MMC-Dcat) and two types of anionic MMC-D (MMC-Dan) with different carrier molecular weights were used. After bolus arterial injection in normal muscles, 83-96% of injected MMC-D was recovered in the venous outflow regardless of the carrier size or charge, whereas less than 60% of MMC was recovered in the same system. By applying statistical moment analysis to the outflow pattern of these drugs, pharmacokinetic parameters representing their disposition characteristics were obtained. Smaller intrinsic clearance (CLint) and distribution volume (V) were noted for MMC-D than for MMC, indicating low extravascular diffusion of MMC-D. In the tumor-bearing muscle, blood contamination from other parts of the body increased and a shortage of flow recovery due to the neovascularization of the tumors occurred. The disposition parameters of MMC-Dcat with a molecular weight of 500,000 (T-500) indicated some tissue distribution and sequestration in the tumor preparation. After constant infusion of [14C]MMC-D (T-500) for 4 h, tissue radioactivity concentrations were determined in various tissues. A higher radioactivity was observed in the viable region of the tumor and the lymph node compared with the normal muscle tissue and the necrotic region of the tumors. 131I-Labeled human serum albumin also gave similar results. In conclusion, higher tumor localization of antitumor agents may be made possible by the application of macromolecular prodrugs.     

Disposition of anticancer drugs after bolus arterial administration in a tissue-isolated tumor perfusion system

Disposition characteristics of various anticancer drugs in a tissue-isolated tumor preparation were studied in Walker 256 carcinosarcoma-bearing rats using an in situ single-pass vascular perfusion technique. Three anticancer drugs, 5-fluorouracil, mitomycin C, and Adriamycin, and two lipophilic prodrugs of mitomycin C were tested in the tumor preparation perfused with Tyrode's solution containing 4.7% bovine serum albumin. After bolus arterial injection of test drugs, their outflow concentration-time curves were analyzed based on statistical moment theory. In each tumor preparation, the injection of drug was paired with that of vascular reference substance, Evans' blue-labeled bovine serum albumin, and disposition parameters of the drug were corrected with those of vascular reference substance. From the mean transit time values of vascular reference substance, the average vascular volume of the tumor preparation was calculated to be 0.063 ml/g, which decreased with tumor growth. All drugs showed significant extraction by the tumor tissue, depending on their physicochemical properties. Distribution volumes of tested drugs were from 1.53 to 3.33 times larger than the vascular volume. Calculated intrinsic clearance values for the protein-unbound fractions increased as the lipophilicity of the drug increased. The potential increase in tumor uptake was observed in lipophilic prodrugs of mitomycin C. The present experimental system is thus suggested to be useful for analyzing drug disposition in tumor tissue.     

Multimodal mass spectrometric imaging of small molecules reveals distinct spatio-molecular signatures in differentially metastatic breast tumor models

Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers.     

Detection of necrosis in human tumour xenografts by proton magnetic resonance imaging.

Tumours with necrotic regions have an inadequate blood supply and are expected to differ from well-vascularised tumours in response to treatment. The purpose of the present work was to investigate whether proton magnetic resonance imaging (MRI) might be used to detect necrotic regions in tumours. MR images and histological sections from individual tumours of three different amelanotic human melanoma xenograft lines (BEX-t, HUX-t, SAX-t) were analysed in pairs. MRI was performed at 1.5 T using two spin-echo pulse sequences, one with a repetition time (TR) of 600 ms and echo times (TEs) of 20, 40, 60 and 80 ms and the other with a TR of 2000 ms and TEs of 20, 40, 60 and 80 ms. Spin-lattice relaxation time (T1), spin-spin relaxation time (T2) and proton density (N0) were calculated for each volume element corresponding to a pixel. Synthetic MR images, pure T1, T2 and N0 images and spin-echo images with chosen values for TR and TE were generated from these data. T1, T2 and N0 distributions of tumour subregions, corresponding to necrotic regions and regions of viable tissue as defined by histological criteria, were also generated. T1 and T2 were significantly shorter in the necrotic regions than in the regions of viable tissue in all tumours. These differences were sufficiently large to allow the generation of synthetic spin-echo images showing clear contrast between necrosis and viable tissue. Maximum contrast was achieved with TRs within the range 2800-4000 ms and TEs within the range 160-200 ms. Necrotic tissue could also be distinguished from viable tissue in pure T1 and T2 images. Consequently, the possibility exists that MRI might be used for detection of necrotic regions in tumours and hence for prediction of tumour treatment response.     

Spin-Lattice Relaxation Time of Inorganic Phosphate in Human Tumor Xenografts Measured in Vivo by 31P-Magnetic Resonance Spectroscopy Influence of oxygen tension

Previous ³¹P-magnetic resonance spectroscopy (³¹P-MRS) studies have suggested that the spin-lattice relaxation time (T₁) of the inorganic phosphate (P_i) resonance is shorter in well-oxygenated than in poorly oxygenated tumors. Amelanotic human melanoma xenografts were therefore subjected to ³¹P-MRS to investigate whether the T₁ of the P_i resonance might be a useful parameter for assessment of tumor oxygenation status. It was searched for possible correlations between the T₁ of the P_i resonance and oxygen tension or parameters closely related to oxygen tension, including ³¹P-MRS tumor energy status and blood supply per viable tumor cell. Oxygen tension, tumor energy status, and blood supply per viable tumor cell decreased with increasing tumor volume. In contrast to previous suggestions, the T₁ of the P_i resonance decreased with increasing tumor volume and decreasing oxygen tension, tumor energy status, and blood supply per viable tumor cell, possibly because the tumors developed necrotic regions concomitantly with the decrease in oxygenation status, resulting in increased concentrations of freely dissolved para-magnetic ions in the tissue. Consequently, the T₁ of the P_i resonance can probably not be utilized to estimate the oxygenation status of tumors, at least not in tumors with necrotic regions.     

Imaging and modulating antisense microdistribution in solid human xenograft tumor models.

PURPOSE: The tumor microenvironment is complex and heterogeneous, populated by tortuous irregular vasculature, hypoxic cells, and necrotic regions. These factors can all contribute to the biodistribution difficulties encountered by most cancer therapeutic agents. Antisense oligodeoxynucleotides (ASO) are a class of therapeutics where limited information is available about their distribution within a solid tumor environment. EXPERIMENTAL DESIGN: To assess ASO distribution, a fluorescein-labeled phosphorothionated ASO based on the G3139 mismatch control was injected systemically (i.v.) into tumor-bearing severe combined immunodeficient mice. Hoechst 33342 was injected i.v. to visualize active vasculature. Unstained sections were imaged through tiled fluorescence stereomicroscopy and then quantitated using novel algorithms. Tumor sections from four human tumor models were examined (CaSki, DU-145, C666-1, and C15) for hypoxia, apoptosis/necrosis, and morphology. RESULTS: For all four tumors, ASO accumulated within regions of hypoxia, necrosis, and apoptosis. Scatter plots of ASO versus active vasculature generated for each individual tumor revealed a consistent pattern of distribution of the ASO within each model. In C666-1 xenografts, the slopes of these scatter plots were significantly reduced from 0.41 to 0.16 when pretreated with the antivascular agent ZD6126 48 h before ASO injection. This was accompanied by the formation of large disseminated necrotic regions in the tumor, along with a 13.1 mmHg reduction in interstitial fluid pressure. CONCLUSIONS: These data suggest the possibility that these algorithms might offer a generalizable and objective methodology to describe the distribution of molecular therapeutic agents within a tumor microenvironment and to quantitatively assess distribution changes in response to combination therapies.     

Transport of molecules in the tumor interstitium: a review

The transport of fluid and solute molecules in the interstitium is governed by the biological and physicochemical properties of the interstitial compartment as well as the physicochemical properties of the test molecule. The composition of the interstitial compartment of neoplastic tissues is significantly different from that of most normal tissues. In general the tumor interstitial compartment is characterized by large interstitial space, high collagen concentration, low proteoglycan and hyaluronate concentrations, high interstitial fluid pressure and flow, absence of anatomically well-defined functioning lymphatic network, high effective interstitial diffusion coefficient of macromolecules, as well as large hydraulic conductivity and interstitial convection compared to most normal tissues. While these factors favor movement of macromolecules in the tumor interstitium, high interstitial pressure and low microvascular pressure may retard extravasation of molecules and cells, especially in large tumors. These differences in transport parameters have major implications in tumor growth and metastases, as well as in tumor detection and treatment.     

Experimental study of tumor detection using 131I-labeled F (ab)2 fragments of monoclonal antibodies to CA 19-9 and CEA (IMACIS-1)

To determine if IMACIS-1 might have the capability of specifically pinpointing an adenocarcinoma in the human lung, xenografts of adenocarcinomatous nude mice that were injected with IMACIS-1 have been evaluated by scintigraphy, and the IMACIS-1 biodistribution in the tissue measured after sacrifice. Results have revealed that scintigraphic images obtained twenty-four hours after the IMACIS-1 injection showed some activity in the area of the tumor. At seventy-two hours after the IMACIS-1 injection, tumoral radioactivity was only seen in mice with a large tumor. At ninety-two hours after injection, the animals were sacrificed and a biodistribution study was performed. The IMACIS-1 uptake was expressed in counts per minute per gram of tissue. The tissue-to-blood uptake ratio in mice with a large tumor was 8.4 in the viable part of the tumor, 27.2 in the necrotic part of the tumor, 3.0 in the liver, and 1.5 in the spleen, respectively. In contrast, the ratio in mice with a small tumor was 3.4 in the viable part and 18.3 in the necrotic part. Immunoperoxidase staining with either the anti CEA or the anti CA 19-9 antibody was strongly visible in the necrotic part of the tumor.     

An enzymatic method for the consistent production of monodispersed viable cell suspensions from human solid tumors

An enzymatic method is described for disaggregation of viable tumor cells from human solid tumors. The enzymatic cocktail consists of 0.1% collagenase, 0.01% hyaluronidase, and 0.002% deoxyribonuclease. After mechanical mincing of the tumor tissue, tumor specimens are dissociated by incubation in the enzymatic cocktail for 12-18 hours at room temperature. In 17 cases of sarcoma, the mean yield was 5 X 10(6) viable cells per gram tumor tissue. Yield was 1 X 10(7) viable cells per gram tumor tissue in 23 cases of gastrointestinal carcinoma. The viabilities of tumor cell suspensions ranged from 50 to 98%, except for low viabilities in four specimens that were grossly composed almost entirely of necrotic tissue. The dissociation procedure is simple and the viable cell yield is sufficient for applications in studies of human cancer immunobiology.     

Tumor Localization and in vivo Antitumor Activity of the Immunoconjugate Composed of Anti-human Colon Cancer Monoclonal Antibody and Mitomycin C-Dextran Conjugate

The tissue distribution and in vivo antitumor activity of a novel monoclonal antibody-mitomycin C conjugate (A7-MMCD) composed of anti-human MAb A7 and MMC-dextran conjugate were investigated using tumor-bearing mice. A7-MMCD was prepared via an anionic dextran intermediate for the purpose of keeping the non-specific uptake by the reticuloendothelial system to a minimum, ¹¹¹n-labeled A7-MMCD showed about a 5-times-greater accumulation in SW1116 (targeted tumor) than in S180 (non-targeted tumor) 48 h after injection, and produced a tumor-to-blood ratio which was 3 times higher in SW1116-bearing mice than in S180-bearing mice 96 h after injection. Accumulations in the liver, spleen, and kidney were also observed to some extent. Pharmacokinetic analysis revealed that A7-MMCD had nearly the same properties in the body as MMCD_an (MMCD with an anionic charge), i.e. those of a negatively charged macromolecule. Both A7-MMCD and MMCD_an had relatively similar tissue uptake rate indices for the liver and spleen. The tumor uptake rate index for SW1116 was about 2.5 times greater than that for S180, and the total amount of ¹¹¹In-A7-MMCD accumulated in SW1116 was calculated to be approximately 5 times greater than the amount in S180. These results indicated that A7-MMCD could achieve site-specific targeting in the body. Furthermore, in the therapeutic experiment using SW1116 implanted subcutaneously, A7-MMCD suppressed tumor growth significantly, compared to free MMC and MMCD_an. These results suggest that in designing an monoclonal antibody-drug conjugate via an intermediary, the physicochemical properties of intermediate macromolecules must also be taken into consideration to obtain a high degree of efficacy in vivo.     

Characterization of a phage display-derived human monoclonal antibody (NHS76) counterpart to chimeric TNT-1 directed against necrotic regions of solid tumors

To eliminate the human anti-mouse antibody (HAMA) response seen in patients treated with murine and chimeric antibodies, fully human monoclonal antibodies (MAbs) are now being developed. Tumor Necrosis Therapy (TNT) is an approach to tumor targeting that utilizes MAbs directed against common intracellular antigens such as nucleic acids, accessible only in necrotic areas of solid tumors. By binding to the necrotic core of tumors, these TNT MAbs can circumvent many of the limitations of MAbs directed against tumor cell surface antigens. Chimeric TNT-1 (chTNT-1) was first developed from the parent murine antibody by genetically engineering the murine variable regions to the human IgG(1) and kappa constant regions. Although the chimeric antibody's behavior was similar to that of the murine version, the 35% murine homology it shares allows for the potential of a HAMA response. A human antibody derived from a phage display library, designated NHS76, has been developed with similar binding characteristics to the TNT-1. To demonstrate that this genetically engineered human counterpart to chTNT-1 has similar pharmacokinetic characteristics, in vivo behavior, and targeting abilities, both antibodies were rigorously tested in parallel. For these studies, biodistribution analysis in LS174T human colon tumor-bearing nude mice was performed to compare the uptake levels in tumor and normal organs. In addition, mouse imaging and autoradiographic studies were conducted to demonstrate positive uptake in necrotic regions of tumor and negative uptake in viable tissues and organs. The results of these studies confirm the comparable nature of both antibodies and provide the necessary preclinical data to show the suitability of NHS76 as an improved product for the therapy of solid tumors in man.     

Sparse initial entrapment of systemically injected Salmonella typhimurium leads to heterogeneous accumulation within tumors

Blood-borne therapeutics, which rely on diffusion and convection for delivery, often do not accumulate in effective concentrations distant from vasculature and are therefore unable to eradicate all cells within a tumor. Motile bacteria have the potential to overcome the diffusion and pressure gradients that prevent passive materials from penetrating into poorly perfused regions of tumors. Here, we test several proposed mechanisms of Salmonella typhimurium accumulation in tumors, including: (a) entrapment in the chaotic vasculature of tumors; (b) attraction to specific tumor microenvironments; and (c) preferential replication within specific microenvironments. After systemic injection of S. typhimurium into tumor-bearing mice, we used intravital microscopy and histological techniques to quantify their interaction with tumor vasculature. Immediately after injection, few S. typhimurium (<4 in 10,000) adhered to tumor vasculature; most remained passively suspended in the blood. Despite this low initial adhesion, approximately 10,000-fold more S. typhimurium accumulated in tumors than any other organ 1 week after the injection, thus demonstrating their specificity. However, within the tumors, we found that most bacteria were located in necrotic tissue as large colonies far (750 micro m) from functional vasculature. Together, these results suggest that S. typhimurium has limited ability to adhere to tumor vasculature and migrate within tumors and only survives in tissue that becomes necrotic. Although S. typhimurium is a promising delivery vehicle because of its tumor specificity, increasing its intra-tumoral motility should improve its therapeutic effectiveness.     

Tumor localization and in vivo antitumor activity of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C-dextran conjugate.

The tissue distribution and in vivo antitumor activity of a novel monoclonal antibody-mitomycin C conjugate (A7-MMCD) composed of anti-human MAb A7 and MMC-dextran conjugate were investigated using tumor-bearing mice. A7-MMCD was prepared via an anionic dextran intermediate for the purpose of keeping the non-specific uptake by the reticuloendothelial system to a minimum. 111In-labeled A7-MMCD showed about a 5-times-greater accumulation in SW1116 (targeted tumor) than in S180 (non-targeted tumor) 48 h after injection, and produced a tumor-to-blood ratio which was 3 times higher in SW1116-bearing mice than in S180-bearing mice 96 h after injection. Accumulations in the liver, spleen, and kidney were also observed to some extent. Pharmacokinetic analysis revealed that A7-MMCD had nearly the same properties in the body as MMCDan (MMCD with an anionic charge), i.e., those of a negatively charged macromolecule. Both A7-MMCD and MMCDan had relatively similar tissue uptake rate indices for the liver and spleen. The tumor uptake rate index for SW1116 was about 2.5 times greater than that for S180, and the total amount of 111In-A7-MMCD accumulated in SW1116 was calculated to be approximately 5 times greater than the amount in S180. These results indicated that A7-MMCD could achieve site-specific targeting in the body. Furthermore, in the therapeutic experiment using SW1116 implanted subcutaneously, A7-MMCD suppressed tumor growth significantly, compared to free MMC and MMCDan. These results suggest that in designing an monoclonal antibody-drug conjugate via an intermediary, the physicochemical properties of intermediate macromolecules must also be taken into consideration to obtain a high degree of efficacy in vivo.     

Some mechanisms involved in cancer cell detachment by necrotic material

Factors influencing cell detachment are thought to play a role in metastasis and invasion. Previous studies on cylinders punched from Walker 256 (W-256) tumors growing in rats showed that detachment of viable cancer cells was facilitated by their proximity to necrotic regions and exposure to a necrotic extract. By the use of pure cultures of W-256 cells, it is shown that necrotic extracts which are rich in lysosomal hydrolases act directly on W-256 cells, facilitating their in vitro detachment. Lysosomal enzymes have previously been shown to facilitate cell detachment. The partial inhibition of this process by hydrocortisone suggests that the extracts also act by labilizing the W-256 cell lysosomes; this enzyme release is also demonstrable by direct assay. Although secondary lysosomes could not be visualized in the W-256 cells, both they and macrophages release lysosomal hydrolases on freeze-thawing, and could both account for the high enzyme content of the necrotic regions, by a process of continuous secretion and/or cell input. Histologic examination of perinecrotic and peripheral regions of tumors showed a higher proportion of macrophages in the former, which is thought to be mainly due to differential trapping of the macrophages. The results emphasize the functional properties of necrotic regions of tumors and the necessity of referring to specific regions of the same tumor when referring to its metastatic and other properties.     

Novel design principles enable specific targeting of imaging and therapeutic agents to necrotic domains in breast tumors

Introduction

Necrosis at the tumor center is a common feature of aggressive breast cancers and has been associated with poor prognosis. It is commonly identified by means of invasive histopathology, which often correlates with morbidity and potential tumor cell dissemination, and limits the reconstruction of the whole necrotic domain. In this study we hypothesized that non covalent association to serum albumin (SA) and covalent binding to ligands for tumor-abundant cell receptors should synergistically drive selective accumulation and prolonged retention of imaging and therapeutic agents in breast tumor necrotic domains enabling in vivo identification, imaging and possibly treatment of such tumors.

Methods

Cyclo-Arg-Gly-Asp-D-Phe-Lys (c(RGDfK)) were conjugated to bacteriochlorophyll-derivatives (Bchl-Ds), previously developed as photodynamic agents, fluorescent probes and metal chelators in our lab. The c(RGDfK) component drives ligation to α_Vβ₃ integrin receptors over-expressed by tumor cells and neo-vessels, and the Bchl-D component associates to SA in a non-covalent manner. STL-6014, a c(RGDfK)-Bchl-D representative, was i.v. injected to CD-1, nude female mice bearing necrotic and non-necrotic human MDA-MB-231-RFP breast cancer tumors. The fluorescence signals of the Bchl-Ds and RFP were monitored over days after treatment, by quantitative whole body imaging and excised tumor/tissue samples derived thereof. Complementary experiments included competitive inhibition of STL-6014 uptake by free c(RGDfK), comparative pharmacokinetics of nonconjugated c(RGDfK) Bchl-D (STL-7012) and of two human serum albumin (HSA) conjugates: HSA-STL-7012 and HSA-STL-6014.

Results

STL-6014 and STL-7012 formed complexes with HSA (HSA/STL-6014, HSA/STL-7012). STL-6014, HSA-STL-7012 and HSA-STL-6014, selectively accumulated at similar rates, in tumor viable regions over the first 8 h post administration. They then migrated into the necrotic tumor domain and presented tumor half lifetimes (T_1/2) in the range of days where T_1/2 for HSA-STL-6014 > STL-6014 > HSA-STL-7012. No accumulation of STL-7012 was observed. Pre-injection of c(RGDfK) excess, prevented the uptake of STL-6014 in the small, but not in the large tumors.

Conclusions

Non-covalent association to SA and covalent binding to c(RGDfK), synergistically enable the accumulation and prolonged retention of Bchl-Ds in the necrotic regions of tumors. These findings provide novel guidelines and strategy for imaging and treatment of necrotic tumors.     

Hypoxia and necrosis in rat 9L glioma and Morris 7777 hepatoma tumors: comparative measurements using EF5 binding and the Eppendorf needle electrode

PURPOSE: The purpose of this study was to assess the presence of tumor hypoxia using two independent techniques: binding of the 2-nitroimidazole EF5 and Eppendorf needle electrode measurements. The distribution of tumor hypoxia was assessed with respect to tumor necrosis in corresponding histological studies. METHODS AND MATERIALS: Each of several rats bearing a subcutaneous 9L glioma or Morris 7777 hepatoma tumor was given EF5 i.v. to a final, whole-body concentration of 100 microM. About 2.5 h later, each rat was anesthetized, and needle electrode measurements were made in the tumor along 1-5 tracks (30-200 individual measurements). At 3 h post-EF5 injection, the tumor was excised and frozen. Frozen sections were analyzed for the presence and distribution of binding of EF5 and necrosis using immunohistochemical techniques followed by staining with hematoxylin and eosin (H&E). The histochemical analysis and electrode readings in similar regions of the tumor were compared. RESULTS: Electrode measurements were taken at 0.4-mm intervals along one-dimensional tracks, whereas EF5 binding measurements from tissue sections contained two-dimensional information at high spatial resolution ( approximately 2.5 micro). The EF5 measurements showed greater spatial heterogeneity than did the electrode measurements. In tumor regions with minimal necrosis, needle tracks with relatively high pO(2) readings were usually found to contain relatively low EF5 binding, and vice versa. Because EF5 binding is inversely related to tissue pO(2), this result was expected. The expected inverse correlation of the two techniques was most disparate in necrotic tumor regions (confirmed by H&E staining), where needle electrode measurements showed low to zero pO(2) values, but little or no EF5 binding was found. CONCLUSION: The two methods compared in this study operate in fundamentally different ways and provide substantially different information. EF5 binding provided detailed spatial information on the distribution of hypoxia in viable tumor tissue. There was no EF5 binding in necrotic tumor tissue because cells in such tissue were unable to metabolize the drug. In contrast, output from the needle electrode method appeared to represent a "track-average" tissue pO(2) and did not distinguish between extreme hypoxia and either macroscopic or microscopic necrosis. At the present time, the importance of tumor necrosis in determining treatment response is unknown. However, our data suggest that the Eppendorf needle electrode technique will overestimate the presence of hypoxia. Both techniques are potentially limited by sampling errors in tumors with heterogeneous distributions of hypoxia.     

In vivo mapping of fractional plasma volume (fpv) and endothelial transfer coefficient (Kps) in solid tumors using a macromolecular contrast agent: correlation with histology and ultrastructure

Contrast-enhanced MRI, immunostaining and electron microscopy were used to detect areas of intense angiogenesis in experimental tumors. This work was also aimed at evaluating the possible effect of the surrounding tissues on tumor microvasculature and at studying the penetration of macromolecules in avascular areas. Human colon carcinoma cells were implanted in subcutaneous tissue of nude mice. Dynamic T(1)-weigthed 3D pulse sequences were acquired before and after administration of Gd-DTPA-albumin to obtain parametric maps of fractional plasma volume (fpv) and transendothelial permeability (Kps). The maps suggested that tumor can be subdivided into 4 zones located in the peripheral rim (zones I-II) or in the core (zones III-IV) of the tumor itself. Significant differences (p<0.001) were found in the values of Kps and fpv of zones I-II with respect to zones III-IV. In the peripheral rim, permeability was significantly higher (p<0.01) in the muscle-peripheral region (zone I) with respect to the skin-peripheral region (zone II). In areas with high Kps, histological and ultrastructural examination revealed clusters of newly formed vessels and signs of intense permeability. Numerous vascular vesicular organs were visible in these areas. In the tumoral core, analysis of the microcirculatory parameters revealed regions with mild permeability (zone III) and regions with negligible permeability (zone IV). These 2 zones were discriminated by the average value of Kps (p<0.05), while their fpv was not significantly different. Upon histological examination, the tumoral core exhibited necrotic areas; CD31 immunocytochemistry exhibited that it was diffusely hypovascularized with large avascular areas. Upon ultrastructural examination, capillaries were rarely visible and exhibited signs of endothelial cell damage. The results suggest that segmentation based on microvascular parameters detects in vivo zones characterized by immunocytochemical and ultrastructural aspects of intense angiogenesis. The finding that a certain amount of contrast agent penetrates in the tumoral core suggests that high oncotic and hydrostatic pressure only partially hinders the penetration of macromolecules.     

131I-chTNT结合外照射治疗实体瘤的实验研究

目的：比较单用^131I-chTNT与^132I-chTNT结合外照射对荷瘤肺癌动物模型肿瘤生长影响的差异。方法：人鼠嵌合抗肿瘤细胞核单抗（chTNT-3）是针对肿瘤坏死组织的新型抗体,具有广谱的抗肿瘤作用,用^131I标记单克隆抗体形成放射性螯合物。24只人肺腺癌荷瘤裸鼠分为4组,分别给予不同的治疗方案后比较肿瘤中24h放射活度比、肿瘤中放射活度比、平均累积吸收剂量以及累积吸收总量。结果：^131I-chTNT联合外照射组,24h肿瘤放射活度比、肿瘤中放射活度比、平均累积吸收剂量以及累积吸收总量较单纯^131I-chTNT组高（P〈0．05）,与对照组相比差异亦有统计学意义。结论：^131I-chTNT结合外照射治疗实体瘤,在给药前行20Gy的外照射组其肿瘤中24h放射活度比、肿瘤放射活度比、平均累积吸收剂量、累积吸收总量高,有助于提高治疗效果。     

Raman molecular imaging of brain frozen tissue sections

Raman spectroscopy provides a molecular signature of the region being studied. It is ideal for neurosurgical applications because it is non-destructive, label-free, not impacted by water concentration, and can map an entire region of tissue. The objective of this paper is to demonstrate the meaningful spatial molecular information provided by Raman spectroscopy for identification of regions of normal brain, necrosis, diffusely infiltrating glioma and solid glioblastoma (GBM). Five frozen section tissues (1 normal, 1 necrotic, 1 GBM, and 2 infiltrating glioma) were mapped in their entirety using a 300-µm-square step size. Smaller regions of interest were also mapped using a 25-µm step size. The relative concentrations of relevant biomolecules were mapped across all tissues and compared with adjacent hematoxylin and eosin-stained sections, allowing identification of normal, GBM, and necrotic regions. Raman peaks and peak ratios mapped included 1003, 1313, 1431, 1585, and 1659 cm^?1. Tissue maps identified boundaries of grey and white matter, necrosis, GBM, and infiltrating tumor. Complementary information, including relative concentration of lipids, protein, nucleic acid, and hemoglobin, was presented in a manner which can be easily adapted for in vivo tissue mapping. Raman spectroscopy can successfully provide label-free imaging of tissue characteristics with high accuracy. It can be translated to a surgical or laboratory tool for rapid, non-destructive imaging of tumor margins.     

Containment of tumor-colonizing bacteria by host neutrophils

Administration of facultative anaerobic bacteria like Salmonella typhimurium, Shigella flexneri, and Escherichia coli to tumor-bearing mice leads to a preferential accumulation and proliferation of the microorganisms within the solid tumor. Until now, all known tumor-targeting bacteria have shown poor dissemination inside the tumors. They accumulate almost exclusively in large necrotic areas and spare a rim of viable tumor cells. Interestingly, the bacteria-containing necrotic region is separated from viable tumor cells by a barrier of host neutrophils that have immigrated into the tumor. We here report that depletion of host neutrophils results in a noticeably higher total number of bacteria in the tumor and that bacteria were now also able to migrate into vital tumor tissue. Most remarkably, an increase in the size of the necrosis was observed, and complete eradication of established tumors could be observed under these conditions. Thus, bacteria-mediated tumor therapy can be amplified by depletion of host neutrophils.