Clonogenic Fraction Research Articles

EXTH-05. TUMOR TREATING FIELDS IN COMBINATION WITH THE TERT-INHIBITOR ERIBULIN HAVE SYNERGISTIC ANTIPROLIFERATIVE EFFECTS ON HUMAN GLIOBLASTOMA CELLS

Abstract OBJECTIVE Combination therapy, a treatment modality that combines two or more therapeutic agents, is a cornerstone of cancer therapy. The optimal combination therapy for Tumor Treating Fields (TTFields) in glioblastoma (GBM) treatment is unknown. The aim of our study was to analyze, the effects of the TERT-inhibitor eribulin in combination with TTFields on human GBM cells. METHODS Human GBM cells of the established cell lines U87, A172 and U251, and two patient-derived cell lines, were treated with eribulin monotherapy, TTFields monotherapy, or both modalities together. After 72 hours of therapy, cell counts were measured and clonogenic assays were performed. Annexin staining and fluorescence-activated cell scanning (FACS) was used to analyze cell death. RESULTS Overall surviving fractions were 39.8±11.0% for eribulin monotherapy, 32.2±23.9% for TTFields monotherapy, and 10.9±9.9% for the combined treatment. Mean observed annexin positive fractions were 11.2±8.2% (control), 28.6±9.7% (eribulin), 34.8±8.1% (TTFields), and 78.1±13.5% (combination), respectively. The mean clonogenic fractions over all cell lines were 25.9±7.8% for eribulin and 46.4±12.9% for TTFields. For the combination therapy, a synergistic effect with a decreased mean of 3.6% clonogenic fractions was observed. CONCLUSION Eribulin increases cell death and reduces clonogenicity our experiments. Additionally, a synergistic effect of the combined treatment of TTFields and eribulin synergistic was observed. Eribulin in combination with TTFields could be a new effective therapy for GBM.

Combination Treatment of Irradiation and Tumor Treating Fields for Human Glioblastoma Cells

Treatment options of glioblastoma (GBM) are limited. Therapy options include tumor resection, radiation therapy, chemotherapy, and the treatment with Tumor Treating Fields (TTFields). The aim of the study was to analyze, for the first time, the effects of the TERT-inhibitor eribulin in combination with TTFields on cell death of human GBM.U87, A172 and U251, and two patient-derived cell lines (447 and 455), were treated with eribulin, TTFields, or both modalities. After 72 hours of therapy, cell counts were measured. Additionally, clonogenic assays were performed. Cell death was analyzed using annexin V staining and fluorescence-activated cell scanning (FACS). The mean differences between cell counts, clonogenic fractions and FACS were evaluated.The mean surviving fractions over all cell lines were 42.6 ± SD % for eribulin, 35.7 ± % for TTFields, and 15.2 ± SD% for the combination. The mean annexin positive fractions over all cell lines were 12.8 ± SD% (control), 30.1 ± SD% (eribulin), 35.3 ± % (TTFields), and 75.5 ± SD% (combination), respectively. The mean clonogenic fractions over all cell lines were 19.1 ± SD% for eribulin and 33.6 ± SD% for TTFields. In the combination therapy, synergistic effects with a decreased mean of 3.2 ± SD% clonogenic fractions were observed.Eribulin is able to increase cell death and to reduce clonogenicity in human GBM cells. For the combined treatment of TTFields and eribulin synergistic effects on cell proliferation were observed. Our data highlight the therapeutic potential of eribulin used in combination with TTFields and offer important insights for possible new effective GBM therapies.M. Stein: None. P. Beusker: None. H. Goett: None. M. Kolodziej: None. E. Uhl: None.

Abstract PO-087: Novel high-throughput screen in a breast cancer cell line to identify potent radiosensitizers

Abstract There is a need for more broadly applicable radiosensitizers independent of disease state and DNA protein repair status. High throughput screens hold potential for identifying new classes of radiosensitizers from libraries of small molecules. Our group has developed an in vitro high throughput screen identifying radiosensitizers using high content imaging. Due to chromatin condensation, G2/M is acknowledged as the most radiosensitive phase of the cell cycle. We therefore dosed 4T1 breast cancer cells with a library of 1430 FDA approved drugs, then using the high content images, assessed ability to stall in G2/M. We initially eliminated drugs with less than 30% viability from consideration to negate toxicity effects on the CCI. With a CCI cutoff of two standard deviations from the mean, we attained a hit rate of 2.8% with 40 hits. The screen identified both known and novel radiosensitizers belonging to previously unidentified classes. This methodology of cell cycle analysis was confirmed by the more classical flow cytometry assay and a selected hit from the screen was assessed for radiosensitizing ability by clonogenic and γH2AX assays, and in vivo studies. Based on the clonogenic survival fractions, the radiation enhancement ratio was found to be >1 at all doses between 2-6 Gy for the selected drug, with the highest values at 4 and 5 Gy of 4.5. Furthermore, we found two 10 µM doses of the novel radiosensitizer paired with two fractions of 2 Gy was sufficient to significantly decrease 4T1 tumor volume growth in BALB/c mice. Citation Format: Madeleine R. Landry, Allison N. DuRoss, Eunseo Choi, Antony Jozic, Dylan Nelson, Conroy Sun. Novel high-throughput screen in a breast cancer cell line to identify potent radiosensitizers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-087.

A local effect model-based interpolation framework for experimental nanoparticle radiosensitisation data

A local effect model (LEM)-based framework capable of interpolating nanoparticle-enhanced photon-irradiated clonogenic cell survival fraction measurements as a function of nanoparticle concentration was developed and experimentally benchmarked for gold nanoparticle (AuNP)-doped bovine aortic endothelial cells (BAECs) under superficial kilovoltage X-ray irradiation. For three different superficial kilovoltage X-ray spectra, the BAEC survival fraction response was predicted for two different AuNP concentrations and compared to experimental data. The ability of the developed framework to predict the cell survival fraction trends is analysed and discussed. This developed framework is intended to fill in the existing gaps of individual cell line response as a function of NP concentration under photon irradiation and assist the scientific community in planning future pre-clinical trials of high Z nanoparticle-enhanced photon radiotherapy.

Inhibition of Fatty Acid Synthase Sensitizes Prostate Cancer Cells to Radiotherapy.

Many common human cancers, including colon, prostate and breast cancer, express high levels of fatty acid synthase compared to normal human tissues. This elevated expression is associated with protection against apoptosis, increased metastasis and poor prognosis. Inhibitors of fatty acid synthase, such as the cerulenin synthetic analog C75, decrease prostate cancer cell proliferation, increase apoptosis and decrease tumor growth in experimental models. Although radiotherapy is widely used in the treatment of prostate cancer patients, the risk of damage to neighboring normal organs limits the radiation dose that can be delivered. In this study, we examined the potential of fatty acid synthase inhibition to sensitize prostate cancer cells to radiotherapy. The efficacy of C75 alone or in combination with X irradiation was examined in monolayers and in multicellular tumor spheroids. Treatment with C75 alone decreased clonogenic survival, an effect that was abrogated by the antioxidant. C75 treatment also delayed spheroid growth in a concentration-dependent manner. The radiosensitizing effect of C75 was indicated by combination index values between 0.65 and 0.71 and the reduced surviving fraction of clonogens, in response to 2 Gy X irradiation, from 0.51 to 0.30 and 0.11 in the presence of 25 and 35 μM C75, respectively. This increased sensitivity to radiation was reduced by the presence of the antioxidant. The C75 treatment also enhanced the spheroid growth delay induced by X irradiation in a supra-additive manner. The level of radiation-induced apoptosis in prostate cancer cells was increased further by C75, which induced cell cycle arrest in the G2/M phase, but only at a concentration greater than that required for radiosensitization. Radiation-induced G2/M blockade was not affected by C75 treatment. These results suggest the potential use of fatty acid synthase inhibition to enhance the efficacy of radiotherapy of prostate carcinoma and that C75-dependent cell cycle arrest is not responsible for its radiosensitizing effect.

Preclinical Antimyeloma Activity of EDO-S101

e218 were substantially minimized after the heat treatment. The heat treatment also suppressed the clonogenic or self-renewal capacity of these MM cells as determined by in vitro colony formation and in vivo tumor formation in SCID mice, suggesting targeting MM progenitors. Further, the Pim inhibitor SMI16a also reduced the SP sizes and the ability of colony formation in RPMI8226 and KMS11 cells. Interestingly, the Pim inhibition in combination with heat treatment enhanced the induction of CHOP, a suicide mediator, while further reducing the protein levels of IRF4 and c-Myc to facilitate MM cell death. These results collectively demonstrated that hyperthermia is able to impair clonogenic drug-resistant fractions of MM cells, which may be augmented in combination with ER stress inducers, such as bortezomib, as well as Pim inhibition. We are now developing superparamagnetic mesoporous nanoparticles, which are able to deliver tumor-selective hyperthermia and drug release. A new strategy with tumor-selective hyperthermia and drug release warrants further study especially in the setting of drugresistant extramedullary plasmacytomas.

Low Dose Total Body Irradiation Combined With Recombinant CD19-Ligand × Soluble TRAIL Fusion Protein is Highly Effective Against Radiation-resistant B-precursor Acute Lymphoblastic Leukemia in Mice

In high-risk remission B-precursor acute lymphoblastic leukemia (BPL) patients, relapse rates have remained high post-hematopoietic stem cell transplantation (HSCT) even after the use of very intensive total body irradiation (TBI)-based conditioning regimens, especially in patients with a high “minimal residual disease” (MRD) burden. New agents capable of killing radiation-resistant BPL cells and selectively augmenting their radiation sensitivity are therefore urgently needed. We report preclinical proof-of-principle that the potency of radiation therapy against BPL can be augmented by combining radiation with recombinant human CD19-Ligand×soluble TRAIL (“CD19L–sTRAIL”) fusion protein. CD19L–sTRAIL consistently killed radiation-resistant primary leukemia cells from BPL patients as well as BPL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. Low dose total body irradiation (TBI) combined with CD19L–sTRAIL was highly effective against (1) xenografted CD19+ radiochemotherapy-resistant human BPL in NOD/SCID (NS) mice challenged with an otherwise invariably fatal dose of xenograft cells derived from relapsed BPL patients as well as (2) radiation-resistant advanced stage CD19+ murine BPL with lymphomatous features in CD22ΔE12xBCR-ABL double transgenic mice. We hypothesize that the incorporation of CD19L–sTRAIL into the pre-transplant TBI regimens of patients with very high-risk BPL will improve their survival outcome after HSCT.

Retroviral-infection increases tumorigenic potential of MDA-MB-231 breast carcinoma cells by expanding an aldehyde dehydrogenase (ALDH1) positive stem-cell like population

Retroviral transformation has been associated with pro-proliferative oncogenic signaling in human cells. The current study demonstrates that transduction of human breast carcinoma cells (MDA-MB231) with LXSN and QCXIP retroviral vectors causes significant increases in growth rate, clonogenic fraction, and aldehyde dehydrogenase-1 positive cells (ALDH1+), which is associated with increased steady-state levels of cancer stem cell populations. Furthermore, this retroviral-induced enhancement of cancer cell growth in vitro was also accompanied by a significant increase in xenograft tumor growth rate in vivo. The retroviral induced increases in cancer cell growth rate were partially inhibited by treatment with 100 U/ml polyethylene glycol-conjugated-(PEG)-superoxide dismutase and/or PEG-catalase. These results show that retroviral infection of MDA-MB231 human breast cancer cells is capable of enhancing cell proliferation and cancer stem cell populations as well as suggesting that modulation of reactive oxygen species-induced pro-survival signaling pathways may be involved in these effects.

CD19-antigen specific nanoscale liposomal formulation of a SYK P-site inhibitor causes apoptotic destruction of human B-precursor leukemia cells.

We report the anti-leukemic potency of a unique biotargeted nanoscale liposomal nanoparticle (LNP) formulation of the spleen tyrosine kinase (SYK) P-site inhibitor C61. C61-loaded LNP were decorated with a murine CD19-specific monoclonal antibody directed against radiation-resistant CD19-receptor positive aggressive B-precursor acute lymphoblastic leukemia (ALL) cells. The biotargeted C61-LNP were more potent than untargeted C61-LNP and consistently caused apoptosis in B-precursor ALL cells. The CD19-directed C61-LNP also destroyed B-precursor ALL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. This unique nanostructural therapeutic modality targeting the SYK-dependent anti-apoptotic blast cell survival machinery shows promise for overcoming the clinical radiochemotherapy resistance of B-precursor ALL cells.

Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia

AbstractWe report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)–dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL.

The Role of Copper in Disulfiram-Induced Toxicity and Radiosensitization of Cancer Cells

Disulfiram has been used for several decades in the treatment of alcoholism. It now shows promise as an anticancer drug and radiosensitizer. Proposed mechanisms of action include the induction of oxidative stress and inhibition of proteasome activity. Our purpose was to determine the potential of disulfiram to enhance the antitumor efficacy of external-beam γ-irradiation and (131)I-metaiodobenzylguanidine ((131)I-MIBG), a radiopharmaceutical used for the therapy of neuroendocrine tumors. The role of copper in disulfiram-induced toxicity was investigated by clonogenic assay after treatment of human SK-N-BE(2c) neuroblastoma and UVW/noradrenaline transporter (NAT) glioma cells. The synergistic interaction between disulfiram and radiotherapy was evaluated by combination-index analysis. Tumor growth delay was determined in vitro using multicellular tumor spheroids and in vivo using human tumor xenografts in athymic mice. Escalating the disulfiram dosage caused a biphasic reduction in the surviving fraction of clonogens. Clonogenic cell kill after treatment with disulfiram concentrations less than 4 μM was copper-dependent, whereas cytotoxicity at concentrations greater than 10 μM was caused by oxidative stress. The cytotoxic effect of disulfiram was maximal when administered with equimolar copper. Likewise, disulfiram radiosensitization of tumor cells was copper-dependent. Furthermore, disulfiram treatment enhanced the toxicity of (131)I-MIBG to spheroids and xenografts expressing the noradrenaline transporter. The results demonstrate that the cytotoxicity of disulfiram was copper-dependent, the molar excess of disulfiram relative to copper resulted in attenuation of disulfiram-mediated cytotoxicity, copper was required for the radiosensitizing activity of disulfiram, and copper-complexed disulfiram enhanced the efficacy not only of external-beam radiation but also of targeted radionuclide therapy in the form of (131)I-MIBG. Therefore, disulfiram may have anticancer potential in combination with radiotherapy.

Monoclonal Antibodies Against Lgr5 Identify Human Colorectal Cancer Stem Cells

In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target Lgr5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence-activated cell sorting-based detection of Lgr5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam+ Lgr5+ cells. Similarly, primary CRC-derived spheroid cultures, known to be enriched for CSCs, contain high levels of Lgr5+ cells, which decrease upon in vitro differentiation of these CSCs. Selection of the Lgr5(high) CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that Lgr5 expression is dependent on the Wnt pathway and show that Lgr5 overexpression induces clonogenic growth. We thus provide evidence that Lgr5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs.

The antiproton cell experiment—do antiprotons offer advantages over other particle beam modalities?

The use of heavy charged particles for cancer therapy has the potential for a significant improvement of the therapeutic window compared to standard X-ray treatments. This is due to the improved energy deposition profile, exhibiting a well-defined peak at a depth in target controllable by the initial energy of the beam. Particles heavier than protons in addition show an increase in biological effectiveness. Compared to protons or heavy ions, antiprotons deposit additional annihilation energy, mostly by low energy recoils, resulting in an increase of dose and also adding a component with high biological effectiveness in the target region. The relative magnitude of the physical energy deposition of antiprotons compared to protons was measured at Low Energy Antiproton Ring (LEAR) by A. Sullivan, but no study of the biological effect had been conducted prior to the Antiproton Cell Experiment (AD-4/ACE) experiment at CERN. The special conditions found at CERN present significant challenges, but also offer unique opportunities. 500 ns pulses of antiprotons are extracted from the Antiproton Decelerator (AD) at 500 MeV/c momentum. Biological cell samples are irradiated and clonogenic survival fractions are measured for various doses. To extract biological efficiency, the physical dose deposition is obtained by Monte-Carlo calculations in conjunction with shot-by-shot monitoring of the incoming beam intensity and profile using a silicon pixel detector. Also imaging of the pions resulting from antiproton annihilations in the target using silicon pixel detector technology to determine the actual range in more complex targets with strong variations in material densities was carried out. The feasibility of this technique using a novel arrangement of the detector was demonstrated. This paper describes the ACE experiment and focuses on the different detector activities within the AD-4/ACE collaboration, explaining the experimental set-up, physical and biological methods used, recent results, and future plans.

Validation of an Automated Procedure to Isolate Human Adipose Tissue–Derived Cells by Using the Sepax®Technology

The stromal vascular fraction of adipose tissue has gained popularity as a source of autologous progenitor cells for tissue engineering and regenerative medicine applications. The aim of this study was to validate a newly developed, automated procedure to isolate adipose-derived mesenchymal stem/stromal cells (ASCs) from adult human lipoaspirates in a closed and clinical-grade device, based on the Sepax(®) technology. Using a total of 11 donors, this procedure was compared with the standard operator-based manual separation in terms of isolation yield, clonogenic fraction, phenotype, and differentiation potential of ASCs. As compared with the manual process, automation resulted in a 62% higher isolation yield, with 2.6±1.2×10(5) nucleated cells per mL of liposuction, and a 24% higher frequency of clonogenic progenitors. The variability in the isolation yield and clonogenicity across different preparations was reduced by 18% and 50%, respectively. The cytofluorimetric profile and in vitro differentiation capacity into mesenchymal lineages were comparable in the cells isolated using the two procedures. The new Sepax-based process thus allows an efficient isolation of ASCs with higher and more reproducible yields than the standard manual procedure, along with minimal operator intervention. These results are expected to facilitate the use of ASCs for clinical purposes, either within an intraoperative setting or in combination with further in vitro cell expansion/cultivation.

Hypofractionation Results in Reduced Tumor Cell Kill Compared to Conventional Fractionation for Tumors With Regions of Hypoxia

Tumor hypoxia has been observed in many human cancers and is associated with treatment failure in radiation therapy. The purpose of this study is to quantify the effect of different radiation fractionation schemes on tumor cell killing, assuming a realistic distribution of tumor oxygenation. A probability density function for the partial pressure of oxygen in a tumor cell population is quantified as a function of radial distance from the capillary wall. Corresponding hypoxia reduction factors for cell killing are determined. The surviving fraction of a tumor consisting of maximally resistant cells, cells at intermediate levels of hypoxia, and normoxic cells is calculated as a function of dose per fraction for an equivalent tumor biological effective dose under normoxic conditions. Increasing hypoxia as a function of distance from blood vessels results in a decrease in tumor cell killing for a typical radiotherapy fractionation scheme by a factor of 10(5) over a distance of 130 μm. For head-and-neck cancer and prostate cancer, the fraction of tumor clonogens killed over a full treatment course decreases by up to a factor of ∼10(3) as the dose per fraction is increased from 2 to 24 Gy and from 2 to 18 Gy, respectively. Hypofractionation of a radiotherapy regimen can result in a significant decrease in tumor cell killing compared to standard fractionation as a result of tumor hypoxia. There is a potential for large errors when calculating alternate fractionations using formalisms that do not account for tumor hypoxia.

γH2AX Expression in Tumors Exposed to Cisplatin and Fractionated Irradiation

Is retention of gammaH2AX foci useful as a biomarker for predicting the response of xenograft tumors to cisplatin with X-ray? Is a similar approach feasible using biopsies from patients with cervical cancer? Mice bearing SiHa, WiDr, or HCT116 xenograft tumors were exposed to cisplatin and/or three daily doses of 2 Gy. Tumors were excised 24 h after treatment and single cells were analyzed for clonogenic fraction and retention of gammaH2AX foci. Tumor biopsies were examined using 47 paraffin-embedded sections from untreated tumors and 24 sections from 8 patients undergoing radiochemotherapy for advanced cancer of the cervix. Residual gammaH2AX measured 24 h after cisplatin injection accurately predicted surviving fraction in SiHa and WiDr xenografts. When a clinically equivalent protocol using cisplatin and fractionated irradiation was employed, the fraction of xenograft cells lacking gammaH2AX ranked survival accurately but underestimated tumor cell kill. Residual gammaH2AX foci were detected in clinical samples; on average, only 25% of tumor nuclei exhibited one or more gammaH2AX foci before treatment and 74% after the start of treatment. gammaH2AX can provide useful information on the response of human tumors to the combination of cisplatin and radiation, but prediction becomes less accurate as more time elapses between treatment and tumor biopsy. Use of residual gammaH2AX as a biomarker for response is feasible when cell survival exceeds approximately 20%, but heterogeneity in endogenous and treatment-induced gammaH2AX must be considered.

Optimization of equivalent uniform dose using the L-curve criterion

Optimization of equivalent uniform dose (EUD) in inverse planning for intensity-modulated radiation therapy (IMRT) prevents variation in radiobiological effect between different radiotherapy treatment plans, which is due to variation in the pattern of dose nonuniformity. For instance, the survival fraction of clonogens would be consistent with the prescription when the optimized EUD is equal to the prescribed EUD. One of the problems in the practical implementation of this approach is that the spatial dose distribution in EUD-based inverse planning would be underdetermined because an unlimited number of nonuniform dose distributions can be computed for a prescribed value of EUD. Together with ill-posedness of the underlying integral equation, this may significantly increase the dose nonuniformity. To optimize EUD and keep dose nonuniformity within reasonable limits, we implemented into an EUD-based objective function an additional criterion which ensures the smoothness of beam intensity functions. This approach is similar to the variational regularization technique which was previously studied for the dose-based least-squares optimization. We show that the variational regularization together with the L-curve criterion for the regularization parameter can significantly reduce dose nonuniformity in EUD-based inverse planning.

Clinical Treatment of Radiotherapy Tissue Damage by Lipoaspirate Transplant: A Healing Process Mediated by Adipose-Derived Adult Stem Cells

There is evidence that stem cells contribute to the restoration of tissue vascularization and organ function. The objective of this study was to assess the presence of adipose-derived adult stem cells left in their natural scaffold in the purified lipoaspirate and to assess the clinical effectiveness of lipoaspirate transplantation in the treatment of radiation side effects. This study was designed beginning with surgical procedures in 2002 and envisaging a continuous patient follow-up to 31 months. Twenty consecutive patients undergoing therapy for side effects of radiation treatment with severe symptoms or irreversible function damage (LENT-SOMA scale grade 3 and 4) were enrolled. Purified autologous lipoaspirates (60 to 120 cc) taken from a healthy donor site were administered by repeated low-invasive computer-assisted injection. Therapy outcomes were assessed by symptoms classification according to the LENT-SOMA scale, cytofluorimetric characterization, and ultrastructural evaluation of targeted tissue. In the isolated stromal vascular fraction of 2 cc of human lipoaspirate, cells with mesenchymal stem cell physical properties and immunophenotype were in average 1.07 +/- 0.5 percent (n = 4), with a clonogenic fraction of 0.139 percent. At least 1.02 x 10(3) colony-forming units-fibroblast were present in each lipoaspirate. Ultrastructure of target tissue systematically exhibited progressive regeneration, including neovessel formation and improved hydration. Clinical outcomes led to a systematic improvement or remission of symptoms in all evaluated patients, including otherwise untreatable patients exhibiting initial irreversible functional damage. This surgical procedure is a low-invasive therapeutic approach for resolving the late side effects of radiotherapy. According to the proposed hypothesis of the ischemic nature of radiolesions, treatment with lipoaspirate transplantation is potentially extended to other forms of microangiopathies.

Human cord blood-derived cells attain neuronal and glial features in vitro.

Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons, astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes - 11% of the total population of differentiating cells.

A simple analytic derivation suggests that prostate cancer α/β ratio is low

Since the publication of Brenner and Hall’s recent paper (1) suggesting that the a/b ratio for prostate cancer is 1.5 Gy, a debate has been propagated through the radiotherapy community (2–5). This surprisingly low value carries implications of more efficient (and perhaps less costly) regimens of external beam radiotherapy (i.e., hypofractionated schedules) and optimal forms of brachytherapy (i.e., high dose rate). Should it prove to be true, such a low ratio would negate the therapeutic advantage of conventional fractionation regimens and call out for clinical trials. There is certainly indirect evidence for a low a/b ratio. The very small proportion of proliferating cells (6, 7), the very long observed doubling times (8, 9), the slow response to irradiation, and the recent interim results of high-dose-rate brachytherapy of varying fractionation (10) are all are consistent with such a low value. The surprise is that this value is as low as, if not even lower than, normal tissue response to irradiation. Can one apply the linear-quadratic (LQ) equation to outcomes of a population of clinically localized prostate cancer after radiotherapy? The LQ equation, although purely phenomenological, has certainly proven to be of great use, both in the study of cultured tumor cells irradiated in vitro, in comparing regimens of different fractionation, and in the study of normal tissue response to radiation. Although issues of interand intratumor heterogeneity are not directly accounted for in the standard LQ formalism, these issues might not necessarily affect the a/b ratio when applied to changes in fraction size (4). Whereas the a term, a direct measure of radiosensitivity, does depend upon heterogeneity (3) and is intimately tied to the number of clonogens, the ratio a/b might not. We present a simple analytical derivation of a/b that is a logical conclusion from the observation that, for clinically localized prostate cancer, external beam and permanent brachytherapy appear to achieve equivalent outcomes. This derivation makes no assumptions and does not attempt to fit models to specific clinical data. For fractionated external-beam irradiation to a total dose De, with dose per fraction d, the LQ survival fraction of clonogens can be written as follows: