The role of strong hypoxia in tumors after treatment in the outcome of bacteriochlorin-based photodynamic therapy

Blood flow and pO₂ changes after vascular-targeted photodynamic therapy (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F₂BMet) as photosensitizer were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F₂BMet generates both singlet oxygen and hydroxyl radicals under near-infrared radiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changed as a result of the disruption of blood vessels by the treatment. A novel noninvasive approach combining electron paramagnetic resonance oximetry and laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO₂ and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO₂, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase in VEGF level 24 h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, whereas V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over 1 year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO₂ compensatory effects and modest tumor inhibition, but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.     

Photodynamic therapy boosts anti-glioma immunity in mice: a dependence on the activities of T cells and complement C3

Photodynamic therapy (PDT) involves the systemic administration of a tumor-specific photosensitizer and local irradiation of visible light, can generate highly cytotoxic molecular species in the tumor and kill malignant cells directly or by shutting down the tumor microvasculature. Collectively data show that anti-tumor immunity is an important mechanism that mediates the PDT-induced tumor-destroying effects in many types of cancers. However, it is unknown whether PDT also promotes anti-tumor immunity in gliomas in the central nervous system (CNS). Here we show that the PDT generates regional and systemic anti-tumor immunity in mice with G422 gliomas in the brain. The infiltration of immune cells and the release of inflammatory factors, such as TNF-α and IFN-γ, are increased in animals with G422 gliomas following PDT when compared with those without receiving PDT. The lymphocytes that are isolated from PDT-treated mice are able to induce anti-tumor immunity in nude mice. The anti-glioma immunity fostered by PDT is inhibited in complement C3 knockout mice and the nude mice indicate the requirement of the activities of complement C3 and T cells. Thus, T cells that produce cytokines, along with complement C3, may play crucial roles in mediating PDT-induced anti-glioma responses.     

A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors

We report the generation of a transgenic Tie2-GFP athymic nude mouse, carrying green fluorescent blood vessels throughout the body. This transgenic mouse is a tool for studies in vascular biology, and is especially of interest for imaging of tumor angiogenesis and the study of anti-angiogenesis strategies in (human) xenografts. Intravital microscopy identified the presence of blood conducting structures that are not lined by endothelial cells. Dedifferentiation of aggressive tumor cells can lead to acquisition of endothelial characteristics. This process of tumor cell plasticity, also referred to as vasculogenic mimicry, has been suggested to contribute to the circulatory system in a tumor. In plastic EW7 Ewing sarcoma tumors in these Tie2-GFP mice, we observed blood flow in both regular blood vessels and non-fluorescent tumor cell-lined channels, visualizing in vivo hemodynamics in vasculogenic channels. These results demonstrate that the transgenic Tie2-GFP athymic mouse model is a valuable tool for vascular biology research.     

Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor

In the present study, the effects of photodynamic therapy (PDT) with verteporfin on tumor blood flow and tumor regrowth were compared as verteporfin distributed in different compartments within the RIF-1 tumor. Tissue distribution of verteporfin was examined by fluorescence microscopy, and blood flow measurements were taken with a laser Doppler system. It was found that, at 15 min after drug administration, when verteporfin was mainly confined within the vasculature, PDT induced a complete arrest of blood flow by 6 h after treatment. PDT treatment at a longer drug-light interval (3 h), which allowed the drug to diffuse to the tumor interstitium, caused significantly less flow decrease, only to 50% of the initial flow in 6 h. A histological study and Hoechst 33342 staining of functional tumor vasculature confirmed the primary vascular damage and the decrease in tumor perfusion. The regrowth rate of tumors treated with 15-min interval PDT was 64% of that of the control group. However, when tumors were treated with 3-h interval PDT, the regrowth rate was not significantly different from that of the control, indicating that only the 15-min interval PDT caused serious damage to the tumor vascular bed. These results support the hypothesis that temporal pharmacokinetic changes in the distribution of the photosensitizer between the tumor parenchyma and blood vessels can significantly alter the tumor target of PDT.     

Effects of homozygosity of the nude (rnu) gene in an inbred strain of rats: studies of lymphoid and non--lymphoid organs in different age groups of nude rats of LEW background at a stage in the gene transfer

Several age groups of nude homozygous rnu/rnu and heterozygous rnu/+ rats of the same genetic background at an early stage of back-crossing (LEW/Mol) were compared as to body and organ weights, histological appearance and cell density of lymphoid organs, haematological values and differential counts of bone marrow and peripheral blood. No thymic tissue was found in the nude animals. 7-week-old nudes were smaller than control animals and had relatively larger non-lymphoid organs and cell-depleted peripheral lymphoid organs. Other age groups showed little difference. Peripheral blood of nude rats showed no signs of lymphopaenia in contrast with the findings in nude mice. The number of thoracic duct lymphocytes was, however, significantly smaller in all age groups of the nude rats, and the bone marrow tended to contain fewer lymphocytes.     

裸鼠肿瘤动物模型VEGF受体表达及其意义

目的　通过免疫组织化学染色了解flt 1与flk 1 KDR(VEGF的两个高亲和受体 )在人肿瘤细胞皮下接种肿瘤动物模型的血管内皮细胞与肿瘤细胞中的表达。方法　取荷瘤裸鼠皮下接种瘤块 ,漂洗、固定、石蜡连续切片 ,进行两种受体相应免疫组化检测。结果　在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中flt 1的阳性率大部分为强阳性或中阳性 ,而只有在荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中flt 1的阳性率为弱阳性 ,在荷人卵巢癌SKOv3裸鼠的肿瘤细胞中flt 1的表达为阴性。相比较而言 ,在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中KDR的阳性率大部分为中阳性或弱阳性 ,并且在荷人肝癌SMMC 772 1裸鼠 ,荷人胃腺癌SPC A1裸鼠 ,荷人高转移肝癌移植瘤裸鼠 ,荷人卵巢癌SKOv3裸鼠的肿瘤细胞中 ,荷人宫颈癌移植瘤裸鼠和荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中 ,KDR表达为阴性。结论　VEGF受体共同表达于肿瘤血管内皮细胞与肿瘤细胞 ,提示了VEGF与VEGF受体结合作用在肿瘤演化中的重要性 ,为靶向于VEGF受体的基因治疗策略选择裸鼠动物模型提供了参考依据     

恶性横纹肌样瘤（MRT）的起源研究——高变异率HeLa细胞致裸鼠产生MRT的实验研究

HeLa细胞KB株、X株、NM20／X株、H株的染色体众数依次为60±3（超二倍体）、62±3（超二倍体）、68±3（超二倍体和亚四倍体）和78±2（亚四倍体）,所占比率分别为72%～76%,69%,52%和40%。在纯化3代的肿瘤阴性对照二倍体猫肾（染色体众数38所占比率80%）和犬肾原代细胞皮下接种裸鼠的致癌／致瘤率分别为0%（0／22）和0%（0／10）,X株HeLa细胞冻融裂解物皮下接种裸鼠产生进行性缩小肿瘤的比率为20%（1／5）的前提下,HeLa细胞KB株、X株、NM20／X株皮下接种裸鼠产生进行性生长恶性肿瘤的比率分别为100%（10／ 10）,100%（25／25）和100%（5／5）,H株细胞皮下接种裸鼠产生恶性肿瘤的比率为50%（5／10）。其中,只有HeLa细胞KB株10～11代（染色体结构畸变率高达20%,出现18%双着丝点和2%断片）以超高数量接种的1组4只裸鼠（0.17ml12.75×10     

Hypericin-mediated photodynamic therapy in combination with Avastin (bevacizumab) improves tumor response by downregulating angiogenic proteins.

Photodynamic therapy (PDT) is a therapeutic modality in which a photosensitizer is locally or systemically administered followed by light irradiation of suitable wavelength to achieve selective tissue damage. In addition, PDT is an oxygen-consuming reaction, that causes hypoxia mediated destruction of tumor vasculature that results in effective treatment. However, the hypoxic condition within tumors can cause stress-related release of angiogenic growth factors and cytokines and this inflammatory response could possibly diminish the efficacy of PDT by promoting tumor regrowth. In such circumstances, PDT effectiveness can be enhanced by combining angiogenesis inhibitors into the treatment regimen. Avastin (bevacizumab), a vascular endothelial growth factor (VEGF) specific monoclonal antibody in combination with chemotherapy is offering hope to patients with metastatic colorectal cancer. In this study we evaluated the combination of hypericin-mediated PDT and Avastin on VEGF levels as well as its effect on overall tumor response. Experiments were conducted on bladder carcinoma xenografts established subcutaneously in Balb/c nude mice. Antibody array, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) were performed to assess VEGF concentrations in the various treatment groups. Our results demonstrated that the targeted therapy by Avastin along with PDT can improve tumor responsiveness in bladder tumor xenografts. Immunostaining showed minimal expression of VEGF in tumors treated with combination therapy of PDT and Avastin. Angiogenic proteins e.g., angiogenin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and interleukins (IL-6 and IL-8) were also found to be downregulated in groups treated with combination therapy.     

Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.     

Brugia pahangi in nude mice: protective immunity to infective larvae is Thy 1.2+ cell dependent and cyclosporin A resistant

Mechanisms of protective immunity to larvae of Brugia pahangi were studied in congenitally athymic nude C3H/HeN mice and their syngeneic heterozygous littermates. An average 11% of subcutaneous larval inocula was recovered from control nudes 28 days after inoculation. No worms were recovered from nude recipients of viable splenic Thy 1.2+ T lymphocytes from heterozygotes which had killed a priming dose of B. pahangi larvae. Primed T lymphocytes, depleted of either Lyt 1.1+ or Lyt 2.1+ cells or incubated with anti-Thy 1.2 monoclonal antibody and complement, failed to protect nude mice against a larval challenge. Nor were primed B lymphocytes depleted by Thy 1.2+ T cell contaminants protective. Treatment with cyclosporin A (CsA) did not increase the numbers of worms recovered from heterozygotes nor did CsA treatment of heterozygous cell donors abolish the ability of primed Thy 1.2+ T lymphocytes to transfer protection to nude mice. IgG but not IgM antibody titres to B. pahangi antigens were depressed in all CsA-treated mice. CsA treatment of nude mice had no direct effect upon development of B. pahangi larvae. These results show that protective immunity to larvae of B. pahangi in mice depends upon small numbers of Thy 1.2+ T cells which are CsA-resistant.     

RIF-1 tumor treatment in anesthetized mice with minimal effects on blood flow and hypoxia

The injectable anesthetic etomidate and a clip that facilitates hyperthermia by water bath immersion (the "Gibbs clip") were evaluated for their suitability with subcutaneous flank RIF-1 tumors in C3H/HeJ mice. For tumors between 100 and 250 mg (mean, 160 mg), etomidate at 40 mg kg-1 ip did not significantly increase the radiobiologic hypoxic fraction (RHF); as calculated from an in vitro assay after treatment in vivo the RHF increased from 0.06 (95% C.I.:0.03-0.13) to 0.08 (0.04-0.16). In contrast, for larger tumors (270-650 mg; mean, 400 mg) etomidate increased the RHF from 0.08 (0.04-0.17) to 0.28 (0.14-0.60). Holding 250-mg-or-less tumors 3-mm laterally away from the flank in an X-ray jig did not significantly reduce tumor blood flow as inferred from the clearance rates of Xe, but the RHF of 0.15 (0.08-0.26) was significantly (P less than 0.05) greater than the RHF in unanesthetized mice, although not the RHF in anesthetized mice. The Gibbs clip, which folded skin around a tumor to enhance thermal conduction from a water bath, did not impair the increase in tumor blood flow in response to the cardiovascular arousal associated with exposure to a hyperthermic stimulus. Intratumor temperature was within 0.25 degrees C of bath temperature 3 min after the tumor and clip were immersed, but only when rectal temperatures were at 37 degrees C or above; tumor blood flow increased intratumor temperature gradients by 0.10 degrees C for each 1.5 degrees C that the body temperature was below 37 degrees C.     

光学成像技术在体研究肿瘤的光动力效应

光动力疗法 (PDT) 已发展成为一种较成熟的肿瘤治疗方法, PDT 诱导的血管损伤是杀死肿瘤的重要机制之一 . 为了在活体肿瘤模型上实时监测 PDT 导致的血管损伤效应,使用稳定高表达绿色荧光蛋白 (GFP) 的人涎腺腺样囊性癌细胞株 (ACC-M-GFP) ,建立了基于鸡胚尿囊膜 (CAM) 的肿瘤模型 . 应用荧光成像技术对肿瘤的生长位置、大小,以及治疗区域进行方便精确的定位;利用激光散斑成像技术,实时监测 CAM 上肿瘤周围血管的血液动力学参数 . 发现不同光动力剂量所导致的血管损伤有显著不同 . 结果表明,荧光标记的鸡胚尿囊膜肿瘤模型为研究 PDT 导致的血管损伤效应提供了良好的实验模型,激光散斑成像技术适用于实时监测 PDT 过程中血管结构、血流速度的变化,由此得出血液灌注率可用以评估 PDT 对肿瘤周围血管的损伤效应 .     

Lymph flow from murine footpad tumors before and after sublethal hyperthermia.

The effect of local hyperthermia (43.5 degrees C for 1 h) on lymph flow from B16-F10 tumor-bearing foot pads of C57BL/6 mice was measured by monitoring the clearance of 99mTc-labeled human serum albumin. The foot was represented by a single-compartment model enabling a quantitative computation of lymphatic flow from the tumor to regional lymph nodes. Lymphatic flow from untreated tumors was 0.0059 +/- 0.0011 ml/min cm3 compared to 0.0118 +/- 0.0027 ml/min cm3 lymphatic flow from tumors immediately following heating. Morphological alterations in tumor blood vessels result in their high vascular permeability. The increase in lymphatic clearance from tumors after sublethal hyperthermia is compatible with the increase in interstitial fluid formation in tumors based on Starling's Law.     

Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment

Background

Broader clinical acceptance of photodynamic therapy is currently hindered by (a) poor depth efficacy, and (b) predisposition towards establishment of an angiogenic environment during the treatment. Improved depth efficacy is being sought by exploiting the NIR tissue transparency window and by photo-activation using two-photon absorption (2PA). Here, we use two-photon activation of PDT sensitizers, untargeted and targeted to SST2 receptors or EGF receptors, to achieve deep tissue treatment.

Methods

Human tumor lines, positive or negative for SST2r expression were used, as well as murine 3LL cells and bovine aortic endothelial cells. Expression of SST2 receptors on cancer cells and tumor vasculature was evaluated in vitro and frozen xenograft sections. PDT effects on tumor blood flow were followed using in vivo scanning after intravenous injection of FITC conjugated dextran 150 K. Dependence of the PDT efficacy on the laser pulse duration was evaluated. Effectiveness of targeting to vascular SST2 receptors was compared to that of EGF receptors, or no targeting.

Results

Tumor vasculature stained for SST2 receptors even in tumors from SST2 receptor negative cell lines, and SST2r targeted PDT led to tumor vascular shutdown. Stretching the pulse from ~ 120 fs to ~ 3 ps led to loss of the PDT efficacy especially at greater depth. PDT targeted to SST2 receptors was much more effective than untargeted PDT or PDT targeted to EGF receptors.

General significance

The use of octreotate to target SST2 receptors expressed on tumor vessels is an excellent approach to PDT with few recurrences and some long term cures.     

Photodynamic therapy of tumors can lead to development of systemic antigen-specific immune response

Background

The mechanism by which the immune system can effectively recognize and destroy tumors is dependent on recognition of tumor antigens. The molecular identity of a number of these antigens has recently been identified and several immunotherapies have explored them as targets. Photodynamic therapy (PDT) is an anti-cancer modality that uses a non-toxic photosensitizer and visible light to produce cytotoxic reactive oxygen species that destroy tumors. PDT has been shown to lead to local destruction of tumors as well as to induction of anti-tumor immune response.

Methodology/Principal Findings

We used a pair of equally lethal BALB/c colon adenocarcinomas, CT26 wild-type (CT26WT) and CT26.CL25 that expressed a tumor antigen, β-galactosidase (β-gal), and we treated them with vascular PDT. All mice bearing antigen-positive, but not antigen-negative tumors were cured and resistant to rechallenge. T lymphocytes isolated from cured mice were able to specifically lyse antigen positive cells and recognize the epitope derived from beta-galactosidase antigen. PDT was capable of destroying distant, untreated, established, antigen-expressing tumors in 70% of the mice. The remaining 30% escaped destruction due to loss of expression of tumor antigen. The PDT anti-tumor effects were completely abrogated in the absence of the adaptive immune response.

Conclusion

Understanding the role of antigen-expression in PDT immune response may allow application of PDT in metastatic as well as localized disease. To the best of our knowledge, this is the first time that PDT has been shown to lead to systemic, antigen- specific anti-tumor immunity.     

Effect of immune reconstitution on resistance to Brugia pahangi in congenitally athymic nude mice

The dichotomy of resistance to Brugia pahangi (Nematoda: Filarioidea) between nonsusceptible, euthymic C3H/HeN mice, heterozygotic for the "nu" gene (+/nu), and susceptible, congenitally-athymic "nude" (nu/nu) C3H/HeN mice, suggests that resistance is thymus-dependent. To test this hypothesis, the effect of syngeneic neonatal thymus grafts and neonatal thymus cell suspensions on recovery of worms at day 40 PI, and responses to Concanavalin A (Con A) were examined in reconstituted nudes. Nude recipients of a thymus graft 7 or 14 wk before subcutaneous inoculation with 50 infective larvae (L3) yielded no worms and responded strongly to Con A. Serum from these mice reacted in two lines of identity with serum from similarly-infected heterozygotes by double radial immunodiffusion against an adult worm saline extract. Nude recipients of a thymus 2 days or 3 wk before inoculation harbored an average of three or two worms, respectively. Intravenous injection of nude recipients with 10(7) or 10(8) neonatal thymus cells seven weeks before inoculation was less effective in conferring resistance to B. pahangi and responsiveness to Con A. Complete resistance to B. pahangi could be adoptively transferred to nude mice by 10(8) spleen cells obtained from infection-primed heterozygotes and injected intravenously on the day of larval inoculation. The same numbers of worms were significantly reduced. less effective when injected 3 wk before inoculation, although numbers of worms were significantly reduced. Passive transfer of primed heterozygote serum, containing high titers of antibodies to adult worm and larval antigens, failed to protect nude recipients against a larval inoculum in the absence of cellular reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of Optical and Power Doppler Ultrasound Imaging for Non-Invasive Evaluation of Arsenic Trioxide as a Vascular Disrupting Agent in Tumors

Small animal imaging provides diverse methods for evaluating tumor growth and acute response to therapy. This study compared the utility of non-invasive optical and ultrasound imaging to monitor growth of three diverse human tumor xenografts (brain U87-luc-mCherry, mammary MCF7-luc-mCherry, and prostate PC3-luc) growing in nude mice. Bioluminescence imaging (BLI), fluorescence imaging (FLI), and Power Doppler ultrasound (PD US) were then applied to examine acute vascular disruption following administration of arsenic trioxide (ATO).During initial tumor growth, strong correlations were found between manual caliper measured tumor volume and FLI intensity, BLI intensity following luciferin injection, and traditional B-mode US. Administration of ATO to established U87 tumors caused significant vascular shutdown within 2 hrs at all doses in the range 5 to 10 mg/kg in a dose dependant manner, as revealed by depressed bioluminescent light emission. At lower doses substantial recovery was seen within 4 hrs. At 8 mg/kg there was >85% reduction in tumor vascular perfusion, which remained depressed after 6 hrs, but showed some recovery after 24 hrs. Similar response was observed in MCF7 and PC3 tumors. Dynamic BLI and PD US each showed similar duration and percent reductions in tumor blood flow, but FLI showed no significant changes during the first 24 hrs.The results provide further evidence for comparable utility of optical and ultrasound imaging for monitoring tumor growth, More specifically, they confirm the utility of BLI and ultrasound imaging as facile assays of the vascular disruption in solid tumors based on ATO as a model agent.     

Incomplete restoration of t-cell reactivity in nude mice after neonatal allogeneic thymus grafting

Grafting an allogeneic neonatal thymus to a congenitally athymic adult nude mouse allows a sufficient generation of φ-positive lymphocytes to bring their relative and absolute numbers close to the values found in normal mice. However, the T-cell characteristic reactivities of thymus-grafted nudes persistently remain low, especially the responsiveness of lymphoid cell suspensions to T-cell mitogens. In a series of thymus-grafted nudes, significant numbers of thymus donor type lymphocytes were found to persist for long periods of time within the grafted thymus, but their persistence could not be directly correlated with the acceptance of skin of same strain origin as the thymus or with responsiveness to mitogens.     

Modified Leukocyte Filter Removes Tumor Cells from the Salvaged Blood

Background

Intraoperative blood salvage, an effective blood conservation strategy, has not been applied in onco-surgery, because of potential malignant cell contamination. In this study we tested effectiveness of a modified leukocyte depletion filter (M-LDF) for removal of tumor cells.

Materials and Methods

The effects of M-LDF and regular LDF on removal of cells (HepG2 cell line) were compared. The safety of M-LDF was tested with blood (collected and washed during onco-surgery), the salvaged blood mixed with tumor cells from the solid tumor of the same patient, or mixed with HepG2 cells (n=30 in each protocol). Cancer cells were identified by flow cytometry, culture and bioassay with and without filtration.

Results

M-LDF removed 5-log of HepG2 and nucleated cells, which was much higher than regular LDF, and cells were destroyed when they passed through M-LDF. Cytokeratin-positive cells in all samples were removed by M-LDF. Invasive growth adherent cells were found in most of unfiltered samples and 67% of the inoculated nude mice developed tumors in LDF-treated sample. Neither adherent cells nor nude mice developed tumors were found in M-LDF-treated samples.

Discussion and Conclusion

Since M-LDF can effectively remove and destroy cancer cells in the salvaged blood, it has great potential for clinical application.