Apoptosis-inducing effects of jujube (Zǎo) seed extracts on human Jurkat leukemia T cells

Background Jujube (Zǎo) seeds exhibited anticancer effects and used in Chinese medicine for many years. This study aims to investigate the apoptosis-inducing effects of seed extracts from eight different cultivated species (‘Apple’, ‘Bombay’, ‘Jumbo’, ‘Kaew’, ‘Nomsod’, ‘Rianthong’, ‘Samros’, and ‘Taiwan’) on human Jurkat leukemia T cells. Methods We evaluated the effects of seed extracts from eight jujube cultivated species on human Jurkat leukemia T cells. The crude seed extracts were prepared sequentially by using water, 95 % ethanol, dichloromethane, ethyl acetate, chloroform or hexane. The antiproliferative effects of the jujube seed extracts relative to that of melphalan were evaluated by neutral red assays. Apoptotic cell death induced by the ethanolic extracts at 1 × IC50 and 2 × IC50 concentrations was demonstrated by DAPI staining, gel electrophoresis, flow cytometry with Annexin V/propidium iodide staining, and caspase-3, -8, and -9 enzyme activities. Results Ethanolic extracts of ‘Taiwan’, ‘Jumbo’, ‘Nomsod’, ‘Rianthong’, ‘Samros’, and ‘Bombay’, significantly inhibited the proliferation of Jurkat cells compared with untreated cells (all P < 0.001), while the extracts of ‘Kaew’ and ‘Apple’ were inactive. The six active extracts preferentially induced apoptotic cell death in a concentration-dependent manner with DNA fragmentation (2 × IC50). Increased caspase-3 activity was detected after treatment with the six extracts. The ‘Taiwan’, ‘Nomsod’, ‘Jumbo’, and ‘Rianthong’ extracts (2 × IC50) induced both the extrinsic and intrinsic apoptosis pathways by increasing caspase-8 and caspase-9 activity, respectively. Alkaloids (Dragendorff’s method) and reducing sugars (Fehling’s test) were mainly identified in the apoptosis-inducing extracts. Conclusions The tested of six active extracts (‘Taiwan’, ‘Jumbo’, ‘Nomsod’, ‘Rianthong’, ‘Samros’ and ‘Bombay’) contained alkaloids or reducing sugars, and induced caspase-dependent apoptosis in human Jurkat leukemia T cells.

Background
Complementary and alternative medicines have been evaluated in addition to chemotherapy, radiation, and surgery in treating cancer. The development of an effective strategy for cancer drug discovery has been outlined [1]. Apoptosis is a major target for chemoprevention and chemotherapy [2]. Jujube (Zǎo) is a functional food with nutritional value that is consumed in Asia, the Mediterranean and the United States [3][4][5][6]. The two extant species include Indian jujube (Ziziphus mauritiana Lam.) and Chinese jujube (Ziziphus jujuba Mill.) [4], and were reported to exhibit sedative, anodyne, pectoral, stomachic, styptic, and tonic effects [7], as well as anticancer, antianxiolytic, antifungal, and antispastic effects [8].
New jujube hybrids are crossbred from the two varieties (Z. jujuba and Z. mauritiana). Presently, many new cultivated varieties are available in Thailand and independently exhibit various biological functions [25][26][27]. Evidence of changes in phytoconstituents between the wild-type plants and new cultivars has been reported [28,29]. The metabolites found in the leaves of new carrot cultivars possessed higher contents of feruloyl acid, quinic acid, malic acid, and leucine, but lower contents of glucose and sucrose than the wild-type (Z. mauritiana) [28]. A new peanut cultivar possessed higher 4α-monomethylsterol contents, but lower 4-desmethylsterol and triterpene alcohol contents than the wild-type plant [29].
This study aims to investigate the apoptosis-inducing effects of seed extracts from eight different jujube cultivated species (' Apple' , 'Bombay' , 'Jumbo' , 'Kaew' , 'Nomsod' , 'Rianthong' , 'Samros' , and 'Taiwan') on human Jurkat leukemia T cells. The anticancer activities were evaluated based on the cytotoxicity and apoptosis-inducing effects on human Jurkat leukemia T cells, relative to melphalan as a positive control. Nuclear morphological changes and caspase-mediated apoptosis were determined. Phytochemical groups in the seed extracts were identified.

Preparation of crude extracts
Eight jujube cultivated species (' Apple' , 'Bombay' , 'Jumbo' , 'Kaew' , 'Nomsod' , 'Rianthong' , 'Samros' and 'Taiwan') were commercially cultivated and authenticated based on fruit and seed morphological characters by Assistant Professor Thaweesak Thitimetharoch, Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University. The jujubes were collected from the local market in Amphoe Muang, Khon Kaen, Thailand, in 2012. The picture vouchers of the specimens (NW-55-2012-NW-62-2012) were deposited at the herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen province, Thailand. The seed characteristics of each jujube cultivated species (five samples for each) are presented in Fig. 1. The seed was separated and dried at ≤55 °C. The seeds were macerated in deionized water (1 g:3 mL) overnight. The water crude extract was collected after being freeze-dried. The remaining residue from water extraction was dried at 55 °C and further macerated with 95 % ethanol, dichloromethane, ethyl acetate, chloroform or hexane, respectively. Each round of solvent extraction lasted 72 h, using different dry residue to solvent ratios (1 g:3 mL). Solvent was removed by a rotary evaporator (IKA ® , Staufen, Germany), resulting in a dry residue. Six types of crude extract were obtained for each cultivars. The crude extracts were stored at −20 °C. The concentrations of 10, 25, 50, 100, 250 and 500 µg/mL were used in the antiproliferation study.

Cell culture
The human Jurkat leukemia T cells were cultured in RPMI 1640 media while the normal African green monkey kidney epithelial cell line (Vero) was cultured in DMEM. Both cell lines were provided by Associate Professor Sahapat Barusrux. Both media were maintained with 10 % fetal bovine serum and 1 % penicillin-streptomycin. Cells were incubated at 37 °C and 5 % CO 2 .

Antiproliferation assay
The antiproliferation test was determined based on modified neutral red assay [30]. A Jurkat cell density of 5 × 10 5 cells/mL and Vero 3 × 10 5 cells/mL were seeded in 96-well plates and maintained at 37 °C for 24 h in humidified 5 % CO 2 . The extracts dissolved in DMSO were pipetted into each well to generate various final concentrations ranging between 10 and 500 µg/mL. The final concentration of DMSO did not exceed 1 % v/v to minimize the cytotoxic effect of DMSO [30]. After treatment with the extracts for 24 h, cells were re-suspended. Neutral red dyes (50 µg/mL) were added to each well. Cells were incubated for another 2 h, then washed with 1 × PBS (pH 7.4) and lysed with 0.33 % HCl in isopropanol. The absorbance of viable cells and dye solution was measured at 537 nm (650 nm reference wavelength) by a microplate reader (TECAN, Grödig, Austria). The untreated cells comprised the control group and cells treated with alkylating drug melphalan are the positive control. A plot of %cell viability and various extract concentrations was used to calculate the IC 50 -which represents the concentration possessing 50 % antiproliferation. The selectivity index (SI) was calculated from the IC 50 of the extracts in normal Vero cells over the Jurkat cancer cells to indicate the cytotoxic selectivity (i.e., safety) of the crude extracts [31].

Detection of apoptosis Nuclear morphological alteration
Morphological changes in cell nuclei undergoing apoptosis was determined by DAPI staining [32]. The stained nuclei were then detected by a fluorescent microscope (Nikon Eclipse 80i, Kanagawa, Japan). Cells were seeded in 24-well plates at a density of 3 × 10 5 cells/mL per well and incubated for 24 h. Cells were treated with 500 µg/ mL extracts and incubated for another 24 h. Then cells were washed with PBS and collected by centrifugation (Daihan Scientific, Seoul, Korea) at 1677g for 5 min. Cells were fixed in ice-cold methanol for 15 min. After the methanol was removed, the cells were incubated for 30 min, at room temperature, in the dark, with 0.3 µg/ mL DAPI. The mixture of PBS to glycerin (at a 1:1 ratio) was added to achieve a 20-µL volume. Cells were wetmounted on a glass slide and observed under a fluorescent microscope by fluorescence filters with an excitation band of 358 nm and an emission of 461 nm. The images of stained nuclei were captured by the NIS-Element AR 3.2 imaging software (Nikon Instruments Inc, NY, USA).

Mode of cell death
Flow cytometry was used to determine various types of cell death including early and late stage apoptosis as well as necrosis. Annexin V-FITC and propidium iodide (Annexin V-FITC apoptosis detection kit, eBiosciences, Inc., San Diego, CA, USA) [33]. Cells were seeded in 24-well plates at a density of 5 × 10 5 cells/ml per well and incubated for 24 h. Cells were treated with the crude extracts at a concentration of 1 × IC 50

Caspases activity
The activity of caspase-3/7, -8 and -9 were evaluated to confirm whether apoptosis was induced by the jujube seed extract(s) and to determine the apoptosis induction pathway.

DNA fragmentation
Late stage apoptotic death mode was confirmed by a DNA fragment assay. Cells (2 × 10 6 cells/mL density) were seeded in 24-well plates and incubated for 24 h. Cells were treated with the jujube seed extracts as described above in the cell death mode assay. Cells were harvested and washed with PBS. After PBS was removed, 300 µL of lysis buffer (FlexiGenen DNA kit; Qiagen, Germany) was added to each well and mixed thoroughly.
After that 150 µL of denaturation buffer and 20 µL of Protease K (10 mg/mL) were added to the reaction mixture. Cells were incubated at 65 °C for 15 min and 600 µL of absolute isopropanol added and thoroughly mixed until the DNA became visible. DNA was collected after centrifugation (Daihan Scientific, Seoul, Korea) at 10,000g for 5 min. The supernatant was discarded and the pellet washed with 70 % ethanol. After the liquid was removed by inverting the Eppendrof tube onto a clean piece of paper, 15 µL of hydration buffer was added to dissolve the DNA for 15 min at 65 °C. The resulting 300 ng/µL of DNA (DNA counting by GE healthcare Life Sciences, United Kingdom) was analyzed by electrophoresis on 1.8 % agarose gels containing 0.75 % ethidium bromide. The DNA was mixed with 6× loading dye and the gel was electrophoresed in 0.5xTBE buffer at 90 V for 1 min and 50 V for 1 h. DNA fragmentation was visualized by a UV transilluminator (Vilberlourmat, Germany) and the image was captured.

Alkylating reaction in vitro
An alkylation reaction of the jujube seed extract was performed to understand the interaction of the extract with DNA resulting DNA damage. The alkylation assay was conducted following the method modified by Machana et al. [30]. NBP (final concentration 20 mM) was prepared in acetonitrile. The buffer solution (pH 4) comprised 0.5 µM of citric acid, 1 µM of boric acid, 4.9 µM of sodium chloride, and 1 M sodium hydroxide adjusted to a volume of 10 mL with deionized water. The mixture of 180 µL NBP and 420 µL buffer solution was prepared and heated to 70 °C for 30 min. The extracts at 2 × IC 50 concentrations dissolved in acetonitrile were pipetted into the NBP mixture. Then, 120 µL of solution mixture was pipetted and added into a 160 µL ethanol-acetonitrile mixture at a ratio of 1:3. The wavelength of NBP adduct was measured at 580 nm at different time points (5,15,30,45, 60 and 75 min).

Phytochemicals screening
Phytochemical screening was performed to identify the presence of the main phytoconstituents in 0.025 g of jujube seed extract of each cultivar. Identification of alkaloids, saponins and carbohydrates were tested as per Anandanayaki [35], while identification of flavonoids was conducted as per Bello et al. [36].

Alkaloids identification
Jujube seed extracts were dissolved in 500 µL of methanol and centrifuged (Daihan Scientific, Seoul, Korea) at 268.3g for 10 min. HCl (1 %, 1 mL) and 1 drop of Dragendorff 's reagent (potassium bismuth iodide) was added to the supernatant. An orange or reddish-brown precipitate indicated a positive test.

Flavonoids identification
Methanol (2.5 mL) and 0.5 mL of 10 % NaOH were added to the extracts. A few drop of 1 % HCl was added to 0.5 ml of supernatant. The disappearance of the yellow color indicated the presence of flavonoids.

Saponins identification
The extracts were diluted with deionized water to a final volume of 1.5 mL and shaken for 30 min. Permanent foamation indicated the presence of saponins.

Reducing sugar identification
Fehling's solution A was prepared by dissolving 3.5 g of copper II sulfate in deionized water and adjusted to 50 mL. Fehling's solution B was prepared by dissolving 17.5 g potassium sodium tartrate with 5 g sodium hydroxide in 50 mL of deionized water. The extracts were diluted in 1.5 mL of deionized water, heated with Fehling's A and B solution (mixture 1:1) for 10 min. An orange-red precipitate indicated a positive result and the presence of reducing sugar.

Statistical analysis
The results from multiple independent experiments were expressed as mean ± standard deviation. Oneway ANOVA was used to test for significant differences between means. P < 0.05 was considered statistically significant. The variation between groups was tested by the Tukey range test with IBM SPSS software version 19.0 (SPSS Inc., USA). The concentration-and time-dependence shown in the graphs was visually determined.

Characteristics of seeds
The eight jujube cultivars had diverse seed characteristics (Fig. 1). Generally, the cultivars had different shapes, sizes, and gnarled seed surfaces, with the average width and length being 0.5 cm and 1.6 cm, respectively. The 'Nomsod' and 'Taiwan' cultivars had the largest seed sizes, while the 'Kaew' cultivar had the smallest.

Antiproliferative effects
The antiproliferative effects of seed extracts from the eight jujube cultivars were evaluated on the leukemic Jurkat cell line, in comparison with the non-cancer Vero cell line, by neutral red assays ( Table 1). The cationic neutral red dye can penetrate through cell membranes and lysosomal membranes to attach to anionic charges in the lysosomal matrix of living cells [37].
The antiproliferative effects were represented as IC 50 values calculated from linear plots between the concentrations and the %cell viability. Extracts with the maximum concentration (500 µg/mL) that inhibited cell viability by <50 % were classified as inactive extracts [30]. Seeds extracted using water, dichloromethane, ethyl acetate, or hexane inhibited Jurkat cell viability by <50 %  50 values of the ethanolic extracts were higher than those of melphalan (i.e., lesser antiproliferative effects) (all P < 0.001), the selectivity index of the ethanolic extracts toward Jurkat cells was higher than that of melphalan. The antiproliferative effects of the ethanolic extracts of the jujube seeds on normal cells were in agreement with previous studies [23,24,38]. Ethanolic extracts of Z. mauritiana seeds showed antiproliferative effects on HL-60, HeLa, and Molt-4 cell lines without affecting the normal HGF cell line [24], and exerted no cytotoxicity on normal rat liver cells [38]. A chloroform extract of Z. jujuba fruit pulp showed antiproliferative effects on MCF-7 and SKBR3 cells without toxicity on normal cells [23]. The present study documented the nonselective cytotoxicity of chloroform seed extracts on Jurkat cells. Owing to the low yields of some chloroform extracts, their cytotoxicity was not determined. Six of the eight ethanolic extracts (i.e., 'Samros' , 'Bombay' , 'Taiwan' , 'Nomsod' , 'Jumbo' , and 'Rianthong') were selected to determine the mode of cell death.

Apoptosis morphological changes
As shown in Fig. 2a, the normal nuclei of untreated cells had an intact round morphology, whereas the apoptotic nuclei of cells treated with 500 µg/mL melphalan showed chromatin condensation with heterogeneous staining (Fig. 2b, c) and apoptotic body formation (Fig. 2d). The nuclear morphologies of Jurkat cells treated with the ethanolic seed extracts (500 µg/mL) for 24 h were shown in Fig. 2e. The six ethanolic jujube seed extracts induced apoptosis with nuclear morphological changes reminiscent of apoptotic characteristics (i.e., cell shrinkage, chromatin condensation, membrane blebbing, and apoptotic body formation).

Mode of cell death
The stages of apoptosis can be differentiated from necrotic cell death by flow cytometry using Annexin V-FITC/propidium iodide (PI) staining of cells. Apoptotic lymphocytes are identified by their externalized phosphatidylserine (PS) bound with Annexin V-FITC (green), while necrotic cells are identified by their DNA intercalation of PI (red) [39]. During the early stages of apoptosis, the PS comprising the cell membrane flip to the outside and bind with Annexin V-FITC, showing a cell dot plot in quadrant 4. Damaged or injured cells lose their membrane integrity, including nuclear membrane integrity, such that PI can pass through the nuclear membrane and appear in quadrant 1 (necrotic cells). In the late stages of apoptosis, cells are double-stained and appear in quadrant 2. Normal cells are not stained and appear in quadrant 3.
The six ethanolic seed extracts induced apoptosis in a concentration-dependent manner. The ranks of the %total apoptosis-inducing effects at the 2 × IC 50 concentrations after 24 h of incubation were in the descending order: 'Bombay' , 'Jumbo' , 'Nomsod' , 'Samros' , 'Rianthong' , melphalan, and 'Taiwan' . Apoptosis in the HL-60 cell line was induced by the ethanolic jujube seed extracts in a concentration-dependent manner, and our findings were in agreement with a previous report [24].

Caspase activity
Apoptosis can be triggered through both extrinsic and intrinsic pathways, involving caspase cascades that act through initiator caspases and executioner caspases [40,41]. The initiators are caspase-8 for the extrinsic pathway and caspase-9 for the intrinsic pathway. When the initiator caspases are activated by apoptotic stimuli, the function of the executioner caspase-3 is triggered, which culminates in DNA fragmentation and apoptotic body formation [2,39,41]. The present study assessed the activity of both initiator caspases, caspase-8 and caspase-9, to distinguish the extrinsic pathway from the intrinsic apoptotic pathway. Executioner caspase-3 activity was also determined. Increased caspase-3 activity was detected at 30 min in Jurkat cells (Fig. 4), suggesting that apoptosis induction by the jujube seed extracts was mediated through the caspase-mediated pathways. 'Taiwan' , 'Nomsod' , 'Jumbo' , 'Rianthong' , and melphalan all significantly increased caspase-8 and caspase-9 activity compared with untreated cells at different time points (all P < 0.001). The increases in caspase-8 activity for 'Taiwan' vs. melphalan differed significantly (P = 0.006). However, there were no significant differences in the increases in caspase-8 activity for 'Samros' and 'Bombay' did not increase the caspase-8 and -9 activities, although they did activate caspase-3 activity. While activation of caspase-9 was not evident, the 'Samros' and 'Bombay' extracts were postulated to cleave the caspase-8 substrate earlier than 15 min because of the high activity at 15 min and lower activities at later time points. Activation of caspase-9 and -3 in a concentration-dependent manner in melanoma cells was previously reported for polysaccharides extracted from the fruit part of Z. jujuba [42]. The ethanolic extracts of 'Taiwan' , 'Jumbo' , 'Nomsod' , 'Rianthong' , 'Samros' , and 'Bombay' induced apoptosis via a caspase-mediated pathway in a similar manner to melphalan, but to different extents. The ethanolic extracts of 'Taiwan' , 'Nomsod' , 'Jumbo' , and 'Rianthong' and melphalan evidently induced apoptosis via the intrinsic and extrinsic pathways.

DNA fragmentation
DNA fragmentation occurs through the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into internucleosomal fragments of 180 bp and multiples thereof. Similar to the case for melphalan, all six ethanolic jujube seed extracts at 2 × IC 50 concentrations clearly exhibited the characteristic DNA ladder formation (Fig. 5).

Alkylation effects of the selected crude extracts compared with melphalan
An alkylation reaction of the jujube seed extracts was performed to understand the interactions of the extracts with DNA resulting in DNA damage, by testing their alkylating activity. The nitrobenzylpyridine (NBP) assay was used as a model for nucleophilic DNA bases [43][44][45]. This reaction depends on high temperature and low acidic media to catalyze NBP, which is converted into a blue/violet chromophore product. This specific reaction is positively detected in the presence of electrophilic functional groups or alkylating agents. Only the alkylating drug melphalan led to NBP product formation, suggesting that the DNA damage induced by the jujube seed extracts did not arise from an alkylation reaction. The findings also implied that no electrophilic functional groups were present in the constituents of the jujube seed extracts.

Phytochemical constituents of the extracts
The phytochemical identification of the ethanolic jujube seed extracts showed that alkaloids predominated in all jujube cultivated species followed by reducing sugars ( Table 2). Neither saponins nor flavonoids were found in the cultivars tested. The ' Apple' , 'Taiwan' , and 'Nomsod' extracts had relatively high amounts of alkaloids, followed by the 'Samros' , 'Bombay' , 'Jumbo' , 'Kaew' , and 'Rianthong' extracts. The 'Nomsod' and 'Rianthong' extracts had relatively high amounts of reducing sugars.
The ethanolic extracts of jujube seeds from different cultivars exhibited diverse inhibition of leukemic Jurkat cell viability. The chloroform extracts and melphalan were toxic toward both Jurkat and Vero cells. The ethanolic extracts selectively caused death in Jurkat cells, but not in normal Vero cells. The 'Taiwan' extract exhibited the highest cytotoxicity followed by the 'Jumbo' , 'Nomsod' , 'Rianthong' , 'Samros' , and 'Bombay' extracts. The modes of cell death induced by these six jujube extracts were distinguished and confirmed by different methods. The Jurkat cells treated with these six ethanolic extracts exhibited various stages of apoptotic nuclear morphological alterations similar to those found in Jurkat cells treated with melphalan. The analyses for mode of cell death at 12 h, detected by flow cytometry, revealed that the 'Bombay' extract at 2 × IC 50 induced significantly more apoptosis than the other extracts and melphalan (99.2 ± 0.4 %; P < 0.001). At 24 h, the 'Bombay' , 'Jumbo' , and 'Nomsod' extracts induced apoptotic cell death at 97.3 ± 2.9 %, 93.7 ± 2.4 %, and 86.0 ± 1.9 %, respectively. Although 'Taiwan' and 'Jumbo' were the first two cultivars possessing high cytotoxicity, they also caused higher degrees of undesirable necrotic cell death. DNA damage was detected in the Jurkat cells treated with the 'Taiwan' , 'Jumbo' , 'Nomsod' , 'Rianthong' , 'Samros' , and 'Bombay' extracts. This DNA damage did not occur via an alkylation reaction between the extracts and the nucleophilic DNA model, as confirmed by the NBP assays. The Jurkat cellular changes, reflecting apoptosis induced by the seed extracts of the six jujube cultivars, were mediated by caspases. Increased caspase-3 activity was detected after all six extract treatments. The 'Taiwan' , 'Nomsod' , 'Jumbo' , and 'Rianthong' extracts induced apoptosis through both extrinsic and intrinsic apoptosis pathways, based on increases in caspase-8 and -9 activities, respectively. The 'Samros' and 'Bombay' extracts seemed to augment the initiator caspase-8 activity at an earlier time point, but did not induce the intrinsic apoptosis pathway.

Conclusions
The six active extracts ('Taiwan' , 'Jumbo' , 'Nomsod' , 'Rianthong' , 'Samros' and 'Bombay') contained alkaloids and reducing sugars, and induced caspase-dependent apoptosis in human Jurkat leukemia T cells. Authors' contributions NW designed the study. NT performed the experiment, collected, and analyzed the data. NW and NT wrote the manuscript. NW, SB and SS revised the manuscript. All authors read and approved the final manuscript.


s2fieldsofstudy: Biology Medicine

text:
p27KIP1 and PTEN cooperate in myeloproliferative neoplasm tumor suppression in mice

PTEN acts as a phosphatase for PIP3 and negatively regulates the PI3K/AKT pathway, and p27KIP1 is a cyclin-dependent kinase inhibitor that regulates the G1 to S-phase transition by binding to and regulating the activity of cyclin-dependent kinases. Genetic alterations of PTEN or CDKN1B (p27KIP1) are common in hematological malignancies. To better understand how mutations in these two genes might cooperate in leukemogenesis, we inactivated both genes in the hematological compartment in mice. Here, we show that the combined inactivation of Pten and Cdkn1b results in a more severe myeloproliferative neoplasm phenotype associated with lower hemoglobin, enlarged spleen and liver, and shorter lifespan compared to inactivation of Pten alone. More severe anemia and increased myeloid infiltration and destruction of the spleen contributed to the earlier death of these mice, and elevated p-AKT, cyclin D1, and cyclin D3 might contribute to the development of this phenotype. In conclusion, PTEN and p27KIP1 cooperate in tumor suppression in the hematological compartment.

Background
PTEN (phosphatase and tension homolog deleted on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 and is one of the most commonly mutated or deleted genes in human cancers, including acute lymphoblastic leukemia, juvenile myelomonocytic leukemia, and non-Hodgkin's lymphoma [1,2]. PTEN acts as a phosphatase for phosphatidylinositol-3,4,5trisphosphate (PIP3) and negatively regulates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway [3]. The CDKN1B gene encodes p27 KIP1 , which belongs to the Cip/Kip family of cyclin-dependent kinase inhibitors. p27 KIP1 is a key regulator of the G1 to S-phase transition by inhibiting cyclinD1/CDK4 and cyclinE/CDK2 complexes [4]. Deletions and other cytogenetic aberrations involving CDKN1B have been reported in a variety of leukemias [5][6][7]. In addition, CDKN1B expression can be a useful prognostic molecular marker for acute myeloid leukemia, where low CDKN1B expression is associated with high proliferation and, therefore, with a favorable response to chemotherapy [6]. Inactivation of the tumorsuppressor gene PTEN and lack of CDKN1B expression have been detected in some kinds of cancer, including most advanced prostate cancers and lymphomas [8,9]. It has been shown that the combined loss of PTEN and p27 KIP1 is associated with tumor cell proliferation and increased risk of recurrent disease in localized prostate cancer [10]. Loss of PTEN expression is more frequent in anaplastic large-cell lymphoma, which strongly correlates with the loss of CDKN1B expression [9].
Targeted disruption of the murine Cdkn1b gene causes a gene dose-dependent increase in animal size without other gross morphologic abnormalities [11], and deletion of Pten in the hematopoietic compartment in mice promotes excessive proliferation of leukemogenic stem cells resulting in the development of myeloproliferative

Open Access
Experimental Hematology & Oncology *Correspondence: meng.liu@gu.se neoplasm (MPN) followed by acute leukemia [12]. In mice, concomitant inactivation of Pten and Cdkn1b accelerates spontaneous neoplastic transformation of prostate cancer [8]. In order to better understand the relation and clinical relevance of these two genes in the pathogenesis of hematological malignancies, we used Cre recombinase to simultaneously inactivate Pten and Cdkn1b in the hematopoietic compartment.

Results and discussion
To determine the impact of combined deficiency of PTEN and p27 KIP1 in the hematopoietic compartment, we injected pI-pC into PCM, PM, CM and Ctrl mice. Consistent with previous studies [13], all PM mice died from MPN by 98 days after pI-pC injections (median survival 62 days), whereas CM and Ctrl mice lived much longer and no MPN phenotype was observed in CM mice. However, the maximum survival of PCM mice was only 30 days (median 24 days; p < 0.001 versus PM; Fig. 1a). Two weeks after pI-pC injections, white blood cell counts were 20.8 × 10 9 cells/L in PCM mice compared with mean counts of 18.3 × 10 9 , 13.9 × 10 9 and 13.6 × 10 9 cells/L for PM, CM and Ctrl mice, respectively (Fig. 1b). However, no morphological changes and no increase in the amounts of immature cells, including myeloblasts, could be detected in the blood and bone marrow in PCM mice compared with the other three groups (Fig. 1c, e). More severe anemia and more architectural disruption of the spleen were observed in PCM mice (Fig. 1d, e). Spleen and liver weights in PCM mice increased by 2.3-5.6 and 1.2-2.4-fold, respectively, compared with PM, CM and Ctrl mice (Fig. 2a, b). Fluorescence-activated cell sorting analysis showed an increased proportion of CD11b+/Gr1+ and LSK [Lineage-negative (lin−), Sca-1+, c-Kit+] cells in the spleen of PCM mice compared to PM, CM and Ctrl mice (p < 0.05; Fig. 2c). Splenocytes from PCM mice produced more colonies compared with the other three groups (Fig. 2d). In bone marrow, there were no differences in the percentage of LSK cells (Fig. 2e). No increased colony formation in PCM mice was observed compared to PM mice when replated, and both groups had more colonies than the Ctrl mice when replated (Fig. 2f ). Taken together, the phenotype in PCM mice is severe MPN rather than acute leukemia based on the criteria for classification of hematopoietic neoplasms in mice [14]. More severe anemia and increased myeloid infiltration and destruction of the spleen likely contributed to the earlier death of PCM mice compared with PM mice.
We performed Western blot analysis to determine the knock-out efficiency and the consequences of inactivating PTEN and p27 KIP1 on downstream molecules. Deficiency of PTEN or p27 KIP1 was observed in the respective knock-out mice (Fig. 2g). It has been shown that PTEN activity leads to the induction of p27 KIP1 , which in turn can negatively regulate the transition through the cell cycle [15]. However, the association between PTEN and p27 KIP1 might be different in different kinds of tissues. A lack of convincing correlation between PTEN and p27 KIP1 has been reported for ovarian carcinomas, indicating the possible existence of p27 KIP1 -independent pathways downstream of PTEN [16]. In our study, we found that expression of p27 KIP1 was reduced in the splenocytes of PM mice. PCM mice had higher phosphorylated AKT compared to PM, CM, and Ctrl mice (Fig. 2g), and cyclin D1 and cyclin D3 expression levels were elevated in PCM and PM mice. However, the levels of phosphorylated ERK1/2 were similar in all groups of mice. Previous studies showed the synergistic activity of PI3K/mTOR and JAK2 signaling pathway in the myeloproliferative neoplasms [17,18], therefore it will be interesting to study the JAK2 activity in PTEN and p27 KIP1 knockout mice model.
In conclusion, our results show that PTEN deficiency can promote tumor progression by a decrease in p27 KIP1 levels in the hematological compartment and that PTEN and p27 KIP1 have a cooperative role in leukemia suppression. In addition, our results show that elevated phosphorylated AKT, cyclin D1, and cyclin D3 might play an important role in the progression of the severe MPN phenotype.

Animal procedures
Mice with conditional Pten fl/fl alleles (designated P) with a mixed genomic background of 129S4/SvJae and C57BL/6J were bred with Cdkn1b fl/fl mice (designated C) to generate PC mice. The mice were housed under controlled environmental conditions with free access to water and food. Illumination was on between 0600 and 1800 h. All mice were monitored daily. Groups of 4-week-old mice were injected with 400 µg polyinosinic-polycytidylic acid (pI-pC; Sigma, St Louis, MO). Blood was taken weekly and analyzed with a hematology analyzer KX-21 (Sysmex Europe, Norderstedt, Hamburg, Germany). Three weeks after injection, groups of mice were sacrificed and their tissues were harvested for further analysis. Mice were euthanized by cervical dislocation after carbon dioxide inhalation. In addition, groups of mice were kept for a survival study. If mice had ruffled fur and become listless or lost more than 10 % of their body weight, they were euthanized. All experimental protocols were approved by the regional ethical committee of the University of Gothenburg, Sweden.


extfieldsofstudy: Biology Medicine

text:
Endogenous GABA controls oligodendrocyte lineage cell number, myelination, and CNS internode length

Adjusting the thickness and internodal length of the myelin sheath is a mechanism for tuning the conduction velocity of axons to match computational needs. Interactions between oligodendrocyte precursor cells (OPCs) and developing axons regulate the formation of myelin around axons. We now show, using organotypic cerebral cortex slices from mice expressing eGFP in Sox10‐positive oligodendrocytes, that endogenously released GABA, acting on GABAA receptors, greatly reduces the number of oligodendrocyte lineage cells. The decrease in oligodendrocyte number correlates with a reduction in the amount of myelination but also an increase in internode length, a parameter previously thought to be set by the axon diameter or to be a property intrinsic to oligodendrocytes. Importantly, while TTX block of neuronal activity had no effect on oligodendrocyte lineage cell number when applied alone, it was able to completely abolish the effect of blocking GABAA receptors, suggesting that control of myelination by endogenous GABA may require a permissive factor to be released from axons. In contrast, block of AMPA/KA receptors had no effect on oligodendrocyte lineage cell number or myelination. These results imply that, during development, GABA can act as a local environmental cue to control myelination and thus influence the conduction velocity of action potentials within the CNS. GLIA 2017;65:309–321

Introduction
B y speeding action potential conduction, myelination of CNS axons by oligodendrocytes increases the brain's cognitive abilities. During development or learning, an adjustment of myelin thickness or internode length may be used to tune the conduction speed of myelinated axons (Fields, 2008;Ull en, 2009). This can promote synchronous neuronal firing (Lang and Rosenbluth, 2003;Sugihara et al., 1993), make impulse propagation time less dependent on the spatial trajectory of the axon transmitting information between areas (Salami et al., 2003), or adjust propagation delays to mediate sound localization (Ford et al., 2015;Jeffress, 1948;McAlpine and Grothe, 2003;Seidl et al., 2010). Magnetic resonance imaging (MRI) reveals changes to white matter microstructure-perhaps reflecting alterations of myelination-when human subjects learn a skilled motor task such as playing the piano (Bengtsson et al., 2005) or juggling (Scholz et al., 2009). Analogous MRI changes, accompanied by elevated myelin basic protein (MBP) expression, are observed in rats trained to grasp food pellets (Sampaio-Baptista et al., 2013), and new myelin production is necessary for mice to become skilled wheel runners (McKenzie et al., 2014).
Together, these studies suggest that adaptive myelination is a normal and essential aspect of neural plasticity.
The proliferation of oligodendrocyte precursor cells (OPCs) must be controlled in order to generate the correct number of oligodendrocytes to ensheath the length of axons requiring to be myelinated. OPC proliferation and myelination are coordinated by communication between the axons to be myelinated and the developing oligodendrocyte lineage cells. This has long been known to involve growth factors, but OPCs also receive excitatory and inhibitory synaptic input mediated by glutamate and GABA (Bergles et al., 2000;Ge et al., 2009;Lin and Bergles, 2004;Karadottir et al., 2005Karadottir et al., , 2008Kukley et al., 2007Kukley et al., , 2008Ziskin et al., 2007;Zonouzi et al., 2015), suggesting that these neurotransmitters may also control oligodendrocyte development and myelination. Glutamate has been suggested to block the proliferation and lineage progression of OPCs (Gallo et al., 1996;Yuan et al., 1998), but also promotes myelin formation (Lundgaard et al., 2013;Wake et al., 2011). Endogenous GABA has been reported to have no effect on OPC development (Gallo et al., 1996;Yuan et al., 1998), but may stimulate OPC migration (Tong et al., 2009) and, by inhibiting neuronal activity, might be expected to decrease myelination (Malone et al., 2013;Sampaio-Baptista et al., 2013). However, during hypoxia, a decrease of GABA A receptor mediated signaling to OPCs increases their proliferation whilst delaying myelination (Zonouzi et al., 2015).
Here we demonstrate a strong effect of endogenous GABA release on oligodendrocyte development in cerebral cortical slices. By acting on GABA A receptors, GABA almost halves the number of OPCs and mature oligodendrocytes produced. Consequently, myelin coverage of axons is decreased. Furthermore, the change in the number of OPCs produced is shown to regulate the myelin sheath internode length, which has previously been postulated to be set solely by the axon diameter (Rushton, 1951) or to be an intrinsic property of the oligodendrocytes carrying out the myelination (Bechler et al., 2015). Thus, GABA release from inhibitory interneurons can tune the conduction speed of CNS axons.

Organotypic Cortical Slices and Myelination Assay
Sox10-lox-GFP-STOP-lox-DTA (called Sox10-GFP below) mice express GFP in oligodendrocyte lineage cells (Kessaris et al., 2006), which allowed us to develop an assay for myelination in organotypic brain slices in which oligodendrocyte lineage cells fluoresce green, and neuronal axons and myelin are identified and quantified with immunofluorescence ( Fig. 1). Using this model we could assess whether changes in myelination reflected changes in the number of oligodendrocyte lineage cells, axonal density or the myelinating activity per oligodendrocyte lineage cell.
Myelination develops over about 2 weeks in these cultured slices (Fig. 1A,E). Initially many oligodendrocyte lineage cells are visible but, as neuronal processes develop, these become fewer in number as a result of cell death, and differentiate so that MBP appears. Larger magnification pictures of the cultures and electron microscope images  reveal that the great majority of the MBP is in compact myelin in close apposition to neurofilamentlabeled processes (only 11% of MBP labeled processes were not clearly wrapping neurofilament labeled axons, and those might be processes connecting internodes to the oligodendrocyte somata, or wrapping axons in which the neurofilament labeling was too weak to see). Thus, very few oligodendrocytes express MBP before they myelinate, and labeling is concentrated in compact myelin around axons.
By counting the number of GFP expressing cells, and using the total fluorescence of secondary antibodies to the NF antibody and to the MBP antibody as measures of the amount of neuronal processes and of myelin present (Rinholm et al., 2011), the progress of myelination in the cultures can be assessed as in Fig. 1E. During the first few days in culture the number of oligodendrocyte lineage cells increases as OPCs proliferate, which is followed by a decrease to below the initial number as cells die. After about a week the number of neuronal processes reaches a plateau, while myelination continues for at least another week. To quantify the amount of myelin per neuronal process, we calculated the ratio of the MBP fluorescence to NF fluorescence. At high magnification, in the centre of the cortex, gaps in the myelin corresponding to ankyrin Gexpressing nodes of Ranvier can be seen (Figs. 1B and 5A), allowing quantification of the number of nodes present and of internode length (Fig. 5F).
Myelination was quantified (with Metamorph or Image J) either by measuring the peak intensity of MBP labeling divided by that of the axon labeling (NF200) in the confocal stack image with the largest intensity in each stack, to obtain a measure of myelination per axon (Rinholm et al., 2011), and then averaging over 2-6 stacks from each slice, or by counting the fraction of axons myelinated in four contiguous regions (Fig. 4I, 30 mm by 25 mm), the overall position of which was chosen randomly. The amount of myelin per length of axon was measured by placing a 3 mm by 3 mm square over the first (from the top left corner) myelinated axon found crossing the top edge of each of the regions in Fig. 4I and integrating the MBP intensity over this 3 lm square area. The internode length of the myelin sheath was measured between two nodes of Ranvier, one at least of which was within a 92 lm by 160 lm region initially imaged, and the internode was followed to its end even if that was outside the initially imaged region (Fig. 5F). These measurements of sheath length are approximately 1 lm shorter than the length that would be measured from the centre of one node to the centre of the next. Measurements of the fraction of axons myelinated, the amount of myelin per length of axon, the number of nodes of Ranvier per field of view, the length of Ranvier nodes and the length of internodes were collected with the experimenter being blind to the experimental condition. When measuring internode length, 80% of internodes remained within one 1.38 lm thick confocal image plane, while 20% extended across 2 adjacent planes in depth. The maximum error this could induce in the derived value for the internode length was 0.14%.

EdU Labeling and Detection
5-ethynyl-2'-deoxyuridine (EdU) (Invitrogen) is a thymidine analogue that is incorporated into the DNA of cells as they undergo DNA replication (Chehrehasa et al., 2009). EdU was added to the organotypic slice medium at a concentration of 10 mM, on DIV4 for 48 h, to quantify the percentage of cells that divide between Days 4 and 6 in vitro. EdU developing was performed immediately following immunocytochemistry, with the Click-iT EdU Alexa Fluor-594 Imaging Kit (Invitrogen). Slices were immersed in Click-iT developing cocktail and incubated for 45 min at 21 C in the dark, according to the manufacturer's instructions. Slices were washed three times in PBS and post-stained with DAPI (300 nM, Molecular Probes) to visualize cell nuclei.

Electron Microscopy
At DIV14 organotypic slices were immersion fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer overnight. All slices were then post-fixed in 1% OsO 4 /0.1 M cacodylate buffer (pH 7.3) at 38C for 2 h before washing in 0.1 M cacodylate buffer (pH 7.3). The slices were dehydrated in a graded ethanolwater series at 38C and infiltrated with Agar 100 resin mix. The slice was then cut perpendicularly to the plane of the slice (in the cortical region where myelination within the organotypic slice is most dense), blocked out, and hardened. Ultra-thin sections were taken on a Reichert Ultracut S microtome. Sections were collected and stained with lead citrate. The sections were imaged using a Joel 1010 transition electron microscope and a Gatan Orius camera.

Cortical OPC Cultures
These were as described by Lundgaard et al. (2013). Briefly, purified oligodendrocyte precursors were obtained using the shake off method of McCarthy and de Vellis (1980) applied to mixed glial cultures that had been cultured for 10 days. They were resuspended in DMEM media with modified SATO serum-free supplement (100 mg/mL BSA, 60 ng/mL progesterone, 16.1 mg/mL putrescine, 5 ng/ mL sodium selenite, 5 mg/mL insulin, 5 mg/mL N-acetyl-L-cysteine, 50 mg/mL holo-transferrin and 1% Pen/Strep) and growth factors (PDGF-aa at 10 ng/mL and FGF-b at 10 ng/mL from Peprotech; UK). OPCs were seeded at a density of 22 3 10 3 cells/cm 2 onto PDL coated glass coverslips yielding a purity of 86 6 2% (n 5 18) NG2 1 cells after 3 days of proliferation (following the 10 days in culture with other glia).

Statistics
Data are shown as mean 6 s.e.m. Unless stated otherwise, Student's 2-tailed t-tests were used. For multiple comparisons, P values were corrected using a procedure equivalent to the Holm-Bonferroni method (for N comparisons in an experiment, the most significant P value is multiplied by N, the 2nd most significant by N-1, the 3rd most significant by N-2, etc.; corrected P values are considered significant if they are less than 0.05). Analysis of variance showed that most variability in the data was between different slices rather than between experiments done on different days, so when pooling data between different experiments we used the number of slices as the number of observations for statistical calculations. Numbers on figure bars show number of slices, except where stated otherwise.

Results
GABA, But Not Glutamate, Regulates the Number of Oligodendrocyte Lineage Cells GABA evokes a current in OPCs (Lin and Bergles, 2004), and will also alter neuronal firing which can influence OPC proliferation and myelination (Gibson et al., 2014). To investigate the role of GABA in regulating oligodendrocyte development we used organotypic brain slices made from the frontal cortex of mice that express GFP in oligodendrocyte lineage cells (see Materials and Methods). Because the slices are able to retain the cyto-architecture found in the cortex for many weeks, they are a good model to study neuron-glial communication while allowing pharmacological manipulation. Fixing slices after different durations in culture, and 312 Volume 65, No. 2 labeling for neurofilament 200 (NF) and myelin basic protein (MBP), allowed us to image neuronal processes, oligodendrocyte lineage cells and myelin, and to monitor the development of compact myelin (Fig. 1).
To test the effect of endogenously released glutamate and GABA on the number of oligodendrocyte lineage cells generated, we included the NMDA receptor blocker MK-801 (50 lM), the AMPA/KA receptor blocker NBQX (25 lM) or the GABA A receptor blocker GABAzine (50 lM; bicuculline was not used because it also blocks K 1 channels: Seutin and Johnson, 1999) in the culture medium from days 3 to 14 in vitro. Blocking NMDA receptors led to a 40 6 12% decrease (P 5 0.02) of the amount of neurofilament labeling in the cultures, probably because neurons need a basal level of NMDA receptor activation to survive (Hardingham and Bading, 2003), which precluded a meaningful analysis of whether NMDA receptors regulate myelination. Deleterious effects on neurons were not observed when blocking AMPA/ KA or GABA A receptors. NBQX, which blocks excitatory synaptic transmission from axons to OPCs (Kukley et al., 2007;Ziskin et al., 2007), had no effect on the number of oligodendrocyte lineage cells present at DIV14 (Fig. 2A,B), nor on the amount of labeling for neurofilament (reduced by 3 6 11%, P 5 0.81) or MBP (increased by 4 6 6%, P 5 0.75). In contrast GABAzine, which blocks inhibitory synaptic transmission, dramatically increased the number of oligodendrocyte lineage cells ( Fig. 2A,B); the increase over 7 independent sets of cultures (each from 2-3 animals) was 1.76 6 0.08 fold (Fig. 2B), implying that endogenous GABA release normally decreases the number of oligodendrocyte lineage cells by a factor of 1/1.76 or 43%.
The larger population of oligodendrocyte lineage cells observed in the presence of GABAzine included more mature oligodendrocytes. Using the combination of Sox10-GFP expression and adenomatous polyposis coli (APC) antibody labeling to define mature oligodendrocytes (Bhat et al., 1996) we found that, by 2 weeks in culture, GABAzine produced a large fractional increase in the number of mature (APCexpressing) oligodendrocytes (increased by 73%) as well as in the number of APC-negative GFP-positive OPCs (increased by 48%, Fig. 2D-G). Thus, endogenous GABA, acting via GABA A receptors, greatly decreases the number of both OPCs and mature oligodendrocytes. Increasing GABA A receptor activation, by applying muscimol (10 lM) (Yuan et al., 1998), decreased the number of oligodendrocyte lineage cells present at DIV14 by 23 6 6% (Fig. 2B). Thus, increases and decreases of GABA A receptor activation bidirectionally alter the number of oligodendrocyte lineage cells.
The increase of the number of oligodendrocyte lineage cells produced by GABAzine did not appear to depend on NKCC1 transporters accumulating Clin the cell to shift positive the reversal potential for GABA A receptors (so that GABA depolarizes OPCs: Lin and Bergles, 2004;Tyzio et al., 2011). Blocking NKCC1 with bumetanide (100 lM, from 3 to 14 days in vitro) had no effect on the change of number of oligodendrocyte lineage cells produced by GABAzine or muscimol (Fig. 2B). Blocking GABA B receptors with 50 lM CGP35348 or activating them with 10 lM baclofen also had no effect on the number of oligodendrocyte lineage cells present (Fig. 2B), unlike a previous report for pure OPC cultures (Luyt et al., 2007).

GABA Regulates the Proliferation and Death of Oligodendrocyte Lineage Cells
GABAzine could increase the number of oligodendrocyte lineage cells either by blocking a GABA-mediated suppression of OPC proliferation, or by blocking GABA-evoked cell death. To investigate this we applied GABAzine from 3-6 days in vitro, i.e. the period in Fig. 1E when OPC proliferation dominates. At day 6, NG2-expressing OPCs were the majority of the oligodendrocyte lineage cells (68.5 6 2.8% in control slices and 74.6 6 2.4% in GABAzine treated slices, not significantly different, P 5 0.11). By DIV6, GABAzine had evoked an increase in the number of oligodendrocyte lineage cells (Fig. 2C) similar to that seen at DIV 14 (Fig. 2B), and applying GABAzine after P6 had much smaller effects (data not shown) demonstrating that the effects of GABA A signaling are exerted mainly at the OPC/early oligodendrocyte stage of development.
Using EdU, we found that GABAzine increased the fraction of OPCs that were dividing between days 4 and 6 in vitro ( Fig. 3A-C). In addition, labeling for apoptotic cell death at day 6 in vitro with antibody to cleaved caspase-3, showed that GABAzine reduced the proportion of SOX10-GFP cells undergoing apoptosis ( Fig. 3D-F). Thus, endogenous GABA release normally suppresses OPC proliferation and increases cell death.
We investigated whether these effects of GABA were directly on OPCs, or mediated by changes of neuronal firing, or involved effects both on OPCs and on neurons. A direct suppressive effect of GABA on the proliferation of neural stem cells, mediated by a PI3K-related kinase (PIKK) and histone H2AX, has been reported (Fernando et al., 2011). If GABA acts in the same way on OPCs then blocking the action of PIKK should mimic the effect of GABAzine in our experiments. However, we found that block of PIKK (using ATM kinase inhibitor, 10 lM) had no effect on the number of oligodendrocyte lineage cells (decreased by 3 6 3% in 3 experiments using a total of 22 slices for each condition, not significant: P 5 0.43).
Blocking neuronal firing by applying 1 lM TTX from Days 3 to 6 in vitro also had no effect on the number of . P values are Holm-Bonferroni corrected for 5 comparisons for Gz, Musc, Bumetanide, CGP, Bac, and for 2 comparisons when comparing Bumetanide1Gz or Bumetanide1Musc with Bumetanide (numbers on bars are total numbers of slices from 12 experiments using two or three animals each). C: Effect of GABAzine from DIV3-6 on number of GFP-expressing cells at DIV6 (numbers on bars are total numbers of slices from 7 experiments using two or three animals each). D: Labeling of oligodendrocyte lineage cells (expressing GFP, green) for the mature oligodendrocyte marker APC (white) and for MBP (red). Top arrowed GFP-expressing cell expresses APC, as well as MBP in its myelinating processes (top thin arrow indicates a primary process linking the arrowed soma to a MBP expressing process), unlike the bottom cell. E: Number of cells expressing GFP and APC in the presence of GABAzine, normalized to the number in control conditions (numbers on bars are total number of slices from 3 experiments using 3 animals each). F: Labeling of oligodendrocyte lineage cells (expressing GFP, green) for APC (white) and for the OPC marker NG2 (red). Unlike the left cell which expresses APC, the right arrowed cell lacks APC and expresses NG2. G: Number of cells expressing GFP but not APC in the presence of GABAzine, normalised to the number in control conditions (numbers on bars are as in E).

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Volume 65, No. 2 OPCs produced (decreased by 2.9%, not significant, P 5 0.7), but blocked the increase in OPC number produced by GABAzine (Fig. 3G, increased by 8.4%, not significant, P 5 0.61). If GABA affects proliferation and cell death solely by acting directly on OPCs, this block could be explained by TTX suppressing action potential evoked release of GABA onto OPCs, or blocking voltage-gated Na 1 channels in OPCs that are activated by the depolarization (Lin and Bergles, 2004) that GABA produces. However, in both these scenarios, TTX alone should produce the same increase in proliferation as GABAzine, but it did not (Fig. 3G). An alternative hypothesis is that the increase in OPC number evoked by GABAzine, and its block by TTX, may be due to GABAzine increasing neuronal action potential firing, leading to the release from neurons of a factor promoting OPC proliferation and decreasing OPC death. However, although the GABA A agonist muscimol (which should reduce neuronal firing) decreased the number of oligodendrocyte lineage cells (Fig.  2B), TTX (which should abolish firing completely) had no effect (Fig. 3G). These results might, however, be explained if there are two effects regulating OPC proliferation: a direct effect of GABA on OPCs which suppresses proliferation (Zonouzi et al., 2015), and release of a factor by active neurons that induces the expression of GABA A receptors or signaling molecules downstream from them, that enables the number of oligodendrocyte lineage cells to be regulated by GABA (see Discussion). In order to determine whether neuronal activity was needed to maintain GABA A receptor expression in OPCs (cf. Arellano et al. (2016) who found that the presence of neurons is needed, but that activity is not), we applied TTX to the organotypic slices. We measured GABA-evoked currents at 264 mV (with E Cl 5-4 mV) in patch-clamped Sox10-GFPexpressing OPCs after incubation in TTX from DIV3 to DIV6, and found that the currents were not significantly reduced (19% decrease, P 5 0.53, n 5 9 with and n 5 9 without TTX), in agreement with the lack of dependence of expression on neuronal spiking seen by Arellano et al. (2016).
We attempted to determine whether GABA has direct effects on OPC proliferation by making cortical OPC cultures and activating GABA A receptors with muscimol. In accordance with a recent publication from the Matute laboratory (Arellano et al., 2016), GABA A receptor mRNA expression was downregulated (by 83% and 78% respectively) for the GABA A receptor a 1 and b 2 subunits in pure OPC cultures without the presence of neurons or neuronalconditioned media (after 10 days in mixed glial culture: see Methods). However, a GABA A receptor mediated current was still observed in these cells (at DIV3 in pure OPC cultures after 10 days in mixed glial culture: see Methods). An outward current of 38 6 8 pA was evoked by 100 lM GABA at 244 mV with E Cl set to 287 mV, implying that GABA A receptors were still functional in the cells. We found that activating GABA A receptors with muscimol (10 lM), blocking them with GABAzine (50 lM) or applying TTX (1 lM), from DIV1 to DIV3 in pure OPC culture, had no effect on the number of proliferating cells, generating a 6.5% increase (P 5 0.41), a 7.6% increase (P 5 0.46), and a 3.5% decrease (P 5 0.98), respectively.
Activation of voltage-gated Na 1 channels in OPCs by the depolarization produced by glutamatergic excitatory synaptic input to OPCs does not contribute to the GABAzineevoked increase in OPC number, because having NBQX present with the GABAzine did not prevent the increase in cell number produced by GABAzine (Fig. 3H).

Endogenous GABA Release Decreases Myelination
In contrast to the effect of GABAzine and muscimol on oligodendrocyte lineage cell number, neurofilament labeling was unaffected by these drugs, implying little effect on the growth of neuronal processes, while total MBP fluorescence was increased by 42 6 19% by GABAzine and decreased by 33 6 7% by muscimol (Fig. 4A-F). As an index of myelination per neuronal process, we normalized the summed fluorescence of the MBP present to the summed fluorescence of the neurofilament present. GABAzine increased this index by 26 6 10%, while muscimol decreased it by 37 6 6% (Fig.  4G,H). The GABAzine-evoked myelination increase was, in part, the result of GABAzine increasing by 25 6 6% the fraction of axons myelinated, without significantly changing the number of axons present (Fig. 4I-K). This implies that endogenous GABA release normally decreases the fraction of axons myelinated by a factor of 1/1.25 or 20%.
By selecting only myelinated axons, and measuring the MBP fluorescence per length of axon (Fig. 4L), we found that GABAzine also increased the amount of myelin labeling per length of axon by 28 6 11% (Fig. 4M), implying that endogenous GABA release normally decreases the amount of myelin per myelinated axon by a factor of 1/1.28 or 22%. This might reflect an increase in myelin thickness, or in axon diameter (measurement of which is inaccurate in our light microscopy images), or both. Thus, endogenous GABA release normally decreases both the fraction of axons myelinated and the amount of myelin per axon.

Endogenous GABA Increases Internode Length
The myelin sheath internode length has previously been postulated to be set solely by the axon diameter (Rushton, 1951) or to be an intrinsic property of the oligodendrocytes carrying out the myelination (Bechler et al., 2015). However, in GABAzine there are more OPCs competing to myelinate the same number of axons. In addition to this being a possible reason why more axons become myelinated, it may result in each oligodendrocyte making shorter internodes. We identified Ranvier nodes using antibody to ankyrin G, or as gaps in the MBP-and GFP-labeling of the myelinating processes of oligodendrocytes (Fig. 5A). GABAzine increased, while muscimol decreased, the number of nodes in each 146 lm square field of view (Fig. 5B). The 47% increase in node density produced by GABAzine is larger than the 25% increase in the number of axons myelinated (Fig. 4K), implying that GABAzine decreases the separation of nodes along axons, i.e. decreases the internode length. GABAzine and muscimol did not alter the lengths of the nodes themselves ( Fig. 5C-E). The distribution of internode lengths differed significantly between control slices and GABAzine-exposed slices (Fig. 5F-G), corresponding to a 13% decrease of mean internode length in GABAzine treated slices (Fig. 5H: the Gaussian fits to the distributions in Fig. 5G predict a 21% decrease), which implies that endogenous GABA release normally increases the mean internode length by a factor of 1/1.13 or 12%. 316 Volume 65, No. 2

Discussion
Our data reveal that the neurotransmitter GABA exercises a major influence on the number of oligodendrocyte lineage cells in situ in cerebral cortical slices. Blocking the effects of GABA on GABA A receptors nearly doubled the number of oligodendrocyte lineage cells ( Fig. 2A,B), increased myelination (Fig. 4G,H) and decreased internode length (Fig. 5G,H). Thus, endogenous GABA release is important in determining the development of these cells (Zonouzi et al., 2015). In contrast, blocking endogenous glutamatergic excitation had no effect on the number of oligodendrocyte lineage cells (Fig. 2B). These observations are in contrast to those of Yuan et al. (1998), who found that glutamate decreased the number of oligodendrocytes in cerebellar slices, while GABA had no effect. The reason for this difference is unclear (although we note that LoTurco et al. (1995) found a suppressive effect of GABA on cortical progenitor cell proliferation, similar to what we find for oligodendrocyte lineage cells). It may imply a difference between the neocortex and the cerebellar cortex in the mechanisms regulating cell proliferation and myelination.
The increase in the number of CC1-expressing mature oligodendrocytes and myelination seen in the presence of GABAzine contrasts with the inhibition of lineage progression reported by Zonouzi et al. (2015) when the GABA A receptor blocker bicuculline was injected in vivo. This could reflect the fact that bicuculline salts have nonspecific actions and, in addition to blocking GABA A receptors, also block Ca 21 -activated K 1 channels (Seutin and Johnson, 1999). The increase of myelination is presumably a result of the increase in the number of oligodendrocyte lineage cells which is produced ( Fig. 3A-F) by an increase of proliferation of OPCs, and also a decrease of cell death (which occurs both at the OPC stage and after differentiation into pre-myelinating

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Volume 65, No. 2 oligodendrocytes: Barres et al., 1992;Trapp et al., 1997). The increased number of OPCs available for myelination ( Fig.  2B,C,G) increases the fraction of axons that become myelinated (Fig. 4K) and decreases internode length, presumably because more OPCs myelinate the same axon ( Fig. 5F-H). Thus, the number of oligodendrocyte lineage cells that are produced and survive-and the resulting myelination-are determined not only by a cell-intrinsic clock and/or the availability of growth factors (Raff et al., 1988;Calver et al., 1998;van Heyningen et al., 2001), but also by locally released GABA. How GABAergic signaling suppresses proliferation and increases cell death remains unclear. We have shown that it does not depend on the reversal potential for GABA-evoked currents being maintained positive to the resting potential by NKCC1, since the effect of GABAzine on oligodendrocyte lineage cell number was unaffected by blocking NKCC1 with bumetanide ( Fig. 2B), and so is unlikely to reflect GABAevoked depolarization initiating a Ca 21 influx through voltage-gated calcium channels (although we cannot rule out the possibility that another mechanism keeps [Cl -] i high). In contrast, Zonouzi et al. (2015) observed that knock-out of NKCC1 had effects similar to the application of bicuculline. We considered the following hypotheses for how GABAzine might increase the number of oligodendrocyte lineage cells.
First we postulated that GABA regulates proliferation solely by acting on oligodendrocyte lineage cells (as suggested by Zonouzi et al., 2015). The increase of OPC number produced by GABAzine (Fig. 2B), would then imply that there is a tonic release of endogenous GABA onto OPCs. The lack of effect of TTX on proliferation (Fig. 3G) would imply that this GABA release is not action potential driven. On this hypothesis, therefore, the GABA would have to be released in an action potential independent manner, perhaps from OPCs or astrocytes, as both synthesize GABA from putrescine using monoamine oxidase B (Barres et al., 1990;Yoon et al., 2014). Inconsistent with this, however, we found that GABAzine had no effect on proliferation in the presence of TTX (Fig. 3G).
We therefore turned to the idea that all of the actions of GABAzine are solely on neurons, with GABAzine increasing spiking and promoting the release of a substance that promotes OPC proliferation. This would be consistent with GABAzine having no effect in TTX (Fig. 3G), but to explain why TTX alone has no effect (Fig. 3G) we would have to also postulate that the spiking rate is very low in the absence of GABAzine. If this were the case, however, the GABA A agonist muscimol (which is expected to decrease the spiking rate like TTX), should also have no effect on OPC proliferation, but in fact it decreases proliferation and myelination (Figs. 2B and 4F).
Thus, the effects of the GABAergic agents and TTX that we have observed apparently cannot be explained solely in terms of an action of GABA solely on OPCs (as postulated by Zonouzi et al., 2015) or solely on neuronal spiking. However, the combination of spiking-induced release of a factor regulating OPC proliferation and a direct suppressive effect of GABA A receptor activation on OPC proliferation might explain the results. The difference in behaviour from the cerebellar OPCs studied by Zonouzi et al. (2015) could reflect the difference of brain area studied.
While the exact mechanisms remain to be defined, it is clear that the development of grey matter OPCs is locally regulated by GABA release (presumably from nearby interneurons: Mangin et al., 2008), which decreases myelination but increases internode length (Figs. 4 and 5). Consequently, interneuron activity can tune the conduction speed of nearby axons, and differences in the local density or activity of interneurons along the path of a set of axons might lead to a spatial variation (Tomassy et al., 2014) of the fraction of axons that become myelinated, or of the internode length in the axons that do become myelinated. A spatial variation of internode length has been reported for axons in the auditory system and is predicted to affect conduction speed (Brill et al., 1977;Ford et al., 2015;Seidl et al., 2010).
Finally, as suggested by recent findings (Zonouzi et al., 2015), it is likely that GABAergic effects on myelination could play a role in pathology. Therapeutic drugs affecting the activity of GABA A receptors might alter CNS myelination if they are administered during the period over which oligodendrocytes develop. Indeed, in rats, the anti-epileptic GABA A agonist phenobarbital decreases myelin formation in rat pups when administered either to the pups or to their mothers before birth (Patsalos and Wiggins, 1982).


extfieldsofstudy: Chemistry Medicine

text:
Impact of adjustable cryogel properties on the performance of prostate cancer cells in 3D

Background Biochemical and physical characteristics of extracellular environment play a key role in assisting cell behavior over different molecular pathways. In this study, we investigated how the presence of chemical binding sites, the pore network and the stiffness of designed scaffolds affected prostate cancer cells. Methods A blend of poly hydroxyethyl methacrylate–alginate–gelatin scaffold was synthesized by cryogelation process using polyethyleneglycol diacrylate (PEGda) and glutaraldehyde as cross linkers. The chemical and mechanical scaffold properties were varied by concentration of gelatin and PEGda, respectively. The pore network was modified by applying different ‘freezing time’. Growth, spheroid formation and localization of androgen receptor (AR) were measured to evaluate cell response within various cryogel types. Results Insufficient porosity in combination with a brittle nature affects cell growth negatively. Spheroid size was reduced by porosity, elasticity as well as by the absence of the cell adhesive motif composed of arginine, glycine und aspartic acid (RGD). Localization of AR indicates its activity and should be under normal culture conditions in the nucleus. But in this study, we could investigate for the first time that AR remains in the cytoplasm when AR positive prostate cancer cells are cultured in scaffolds without RGD as well as in case of an insufficient pore network (total porosity under 10 %) and a too less stiffness of around 10 kPa. Conclusions The results indicate that for getting a reliable preclinical drug screening a three-dimensional prostate model system with appropriate biochemical and physical surrounding is needed.

in vitro matrix (Ravi et al. 2015). In general sophisticated 3D tumor spheroids have proven particularly useful for basic cancer research, such as prostate cancer, PrCa (i.e., prostaspheres (Hedlund et al. 1999;Takagi et al. 2007) to mimic the cellular in vivo environment associated with tumor growth (Thom et al. 2014;Kunz-Schughart et al. 2004). Those cell aggregates have significant potential to advance our understanding of cancer progression (Sutherland et al. 1971). Spheroids can be studied in scaffold-free suspension (e.g., 'hanging drops' of medium) or in scaffold-based matrices of different origin.
Among scaffold-based models are polymeric gel matrices of various materials (natural or synthetic origin) or material combinations (Nyga et al. 2011;Fischbach et al. 2007) and purified extracellular matrix (ECM) gels (e.g., Matrigel) (Streuli et al. 1991). Nevertheless, matrigel has batch-to-batch variations due to the different amounts of natural components and a very low elasticity of only 0.5 kPa, which is outside the range of certain cancer types including prostate cancer (65-126 kPa) (Soofia et al. 2009;Wells 2008). Several groups have already reported that biophysical cues of cellular microenvironment in vitro can influence the results in 3D culture, especially spheroid size (Cheng et al. 2013). Thus, the ability to control certain properties (e.g., structure, porosity as well as mechanical stimulation) is an essential parameter for engineering proper scaffolds.
Cryogels, as a type of macroporous hydrogel, synthesized at subzero temperatures, can be applied as 3D structures of different formats like fibres, meshes or sponges and offer the aforementioned controllability (Kirsebom and Mattiasson 2011). The porosity as main result of the cryotropic gelation process appears due to the existence of the solvent crystals acting as porogens for the formation of the pores (Zhao et al. 2011). The outstanding characteristics of cryogels prompted us to look for the scaffold properties and the cellular answer regarding to it to develop a suitable microenvironment for prostate cancer study in vitro. It is well-known that the synthesis parameters for cryogels are crucial for the main characteristics. Nevertheless, it is still less known about the influence on cell culture caused by physical and chemical properties of designed scaffolds.
Thus, here we report the synthesis and characterization of a polyhydroxyethyl methacrylate-alginate-gelatin-poly(ethylene glycol) diacrylate (pHAG-PEGda) cryogel combinations and their effects on culturing the epithelial prostate cancer cell line LNCaP. The polymer combination was chosen based upon the given criteria of the applied components. Gelatin has further well-known cell-adhesive properties by containing the RGD (arginine, glycine und aspartic acid) sequence, which should improve cell attachment (Aplin et al. 1998;Yamada 1991). The synthetic and water-soluble monomer poly-hydroxyethyl methacrylate (HEMA) is also highly biocompatible and is a wellknown material of contact lenses (Fornasiero et al. 2005).
The aim of the present work was to investigate the effects of single physical and chemical characteristics of 3D matrices on culturing prostate cancer cells. Various changes in synthesis protocol offer the possibility to control presence of chemical attractants, like RGD sequence by use of gelatin, establishment of pore network by freezing time and degree of elasticity by cross linker concentration independently of each other. The behavior of LNCaP cells was evaluated by analyzing proliferation rate, spheroid formation and size as well as androgen receptor (AR) localization. This study can be of high importance for the establishment of a suitable 3D platform for preclinical anti-AR drug screening due to the fact that properties of the surrounding scaffold was determined to affect AR localization.

Preparation of pHAG-PEGda cryogels
Different concentrations of poly-hydroxyethyl methacrylate (pHEMA), gelatin, alginates and poly(ethylene glycol) diacrylate (PEGda) were used to synthesize the poly-hydroxyethyl methacrylate-alginate-gelatin-poly(ethylene glycol) diacrylate (pHAG-PEGda) cryogels in water as solvent. The gels were prepared by mixing gelatin and alginate in double-distilled water. The monomer HEMA and the cross linker PEGda were added to the reaction mixture after complete dissolution of the previous polymers. The gel reaction formation was initiated by adding APS/TEMED (0.5 %/0.1 % w/v) and glutaraldehyde (25 % v/v) as gelatin crosslinker. The mixture was poured immediately into 3 ml syringe and frozen overnight (otherwise it will be mentioned in the text) at −21 °C. The polymerized cryogels were cutted into 3 mm thick disks with a diameter of 8 mm, thawed at room temperature and stored in double-distilled water at 4 °C prior to use.

Microstructure analysis using scanning electron microscopy (SEM)
The pore network morphology of cryogels was studied using scanning electron microscope (SEM, Zeiss 60VP). The scaffolds were dried over 2 days at room temperature to remove moisture totally before silver coating was performed using a sputter coater (EMITECH K575X). The microscope was operated at high vacuum of 3.00 kV. If cells were integrated in scaffolds after certain time of cell culture, the samples were first fixed with 2.5 % glutaraldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.4) and dried at least 2 days at room temperature.

Visco-elastic behavior of scaffolds
Compression tests were carried out on RPMI medium saturated cryogels (disks) without cells. Uniaxial stress was given directly to the cylindrical samples using Zwick/Roell Z0.5 machine (Germany) with 50 N load cell by a constant linear compression rate of 1.0 mm/ min. Here the elastic modulus of the cryogels was calculated from the linear region of the stress-strain curve at low strain regions. All measurements were made five times.

Porosity analysis
Mercury intrusion porosimetry (MIP) is a well-known technique used to measure pore size distribution in a porous material. The pore size distribution, pore surface area and pore volume in a cryogel samples was determined using the mercury porosimetry machine (Thermo Finnigan, Pascal 140/440) (Woerly et al. 1998;Lv et al. 2005). The samples were placed into penetrometers which are weighed in front of the experiment. The analysis is based on the principle that a non-wetting liquid (mercury) penetrates into the porous network only under pressure. To determine the porosity, it was assumed that the shape of the pores was cylindrical. The contact angle of mercury is 130°, and the surface tension of mercury is 0.485 N m −1 . The pressure applied is inverse proportional to the inner width of the pore aperture.
The pore diameter was calculated using the Washburn equation: where P is the applied pressure, θ the contact angle and ɣ the surface tension of mercury. The intrusion volume was recorded for pressure ranging from 0 to 480 MPa. The porosity was calculated using the equation: where ds is the skeletal density of the dried gel (derived from gel volume with exclusion of the volume of the sample's pore structure) and db is the bulk density of the dried gel (derived from the gel volume that includes the volume of the sample's pore structure).

Cell culture and proliferation
Human prostate epithelial cancer cells (LNCaP) with cell suspension (2.5 × 105 cells/ µl) were seeded onto sterilized pHAG-PEGda cryogels. The samples were pre-sterilized with RPMI medium supplements with 10 % FCS and 1 % Antibiotic Antimycotic Solution followed by repeated washing with sterilized PBS (pH 7.4). After 2 h of initial cell attachment, the samples were transferred to 6 well culture plates, containing medium and incubated at 37 °C in a humidified 5 % CO 2 atmosphere. Fresh medium was replenished every 2 days. At regular time intervals the cryogels were fixed with 2.5 % glutaraldehyde for scanning electron microscopy analysis. Or in case of paraffin sections with subsequent haematoxylin and eosin (H&E) staining the samples were fixed with 3.7 % formalin. The total metabolic activity of the cells was monitored using CellTiter-Glo ® 3D (Promega). The CellTiter-Glo ® 3D Viability Assay is a homogenous method to determine the number of viable cells in 3D cell culture model systems. The assay based on quantitation of the ATP, which is a marker for the presence of metabolically active cells. In brief, the media was removed from the cell-seeded cryogels and followed by cooling down at room temperature for 30 min. The ready-to-use reagent as well as fresh medium was added with the equal volume to the samples. The measured amount of adenosine triphosphate (ATP) is proportional to the amount of DNA.

Haematoxylin/eosin (H&E) and immunofluorescence (IF) staining
For H&E staining, the reaped cryogels were fixed in 3.7 % formalin solution, dehydrated and embedded in paraffin. Several sections (5 µm thick) were deparaffinized. The deparaffinized sections were subsequently stained by hematoxylin which reacts like a basic dye with a purple-blue color and eosin an acidic dye that is typically reddish or pink. Hematoxylin stains acidic, or basophilic, structure including the cell nucleus. In contrast to that eosin stains basic, or acidophilic, structures including the cytoplasm, cell walls, and extracellular fibers (Fischbach et al. 2007). After finishing the staining, the sections were covered with a cover glass (to make the preparation permanent). For IF, the deparaffinized sections were washed three times PBS (pH 7.4). The nonspecific binding sites were blocked with block buffer (including 10 % goat serum, 0.1 % Tween 20) for 1 h and the sections were then incubated for 120 min at room temperature in mouse anti-Androgen Receptor primary antibody (Dianova DLN-09477) at 1:25 in humid environment. After rinsing with PBS (three times), the sections were incubated for an additional 1 h in goat-anti-mouse Cy3 at 1:200 in humid environment. The sections were counterstained with DAPI at 1:5000 and examined under an inverted fluorescence microscope (Leica DM5500).

Statistical analysis
All quantitative measurements were performed on at least triplicates. All values are expressed as means ± standard deviations (SD). One-way ANOVA with post hoc Tukey tests were used to compare treatment groups in the quantitative measurements and p < 0.05 was used to assess statistical significance.

Physical characterization of different pHAG-PEGda cryogels
Cryogels were synthesized at −21 °C and concentrations of components applied as mentioned in Fig. 1. Under standard synthesis conditions a cryogel with 50 % total porosity, an average pore size of 20 µm and a stiffness of 38 kPa was generated (Fig. 2a). The absence of gelatin (approach 1) has no influence on porosity and elasticity, respectively and only a small reduction of the average pore size (Fig. 2b). Varying freezingtime (approach 2) has a considerable effect on the architectural features of the cryogel. A cryogelation time of only 1 h, leads to less mechanical stability and a brittle nature, which was confirmed by an E modulus of only 10 kPa (Fig. 2c). However, elasticity of the standard cryogel (around 40 kPa) is already reached at 5 h and thus further elongation of freezing time remains without any effect. In comparison to that the effect of freezing time on porosity as well as average pore size is clearly detectable by a stepwise increase of these parameters according to elongating the freezing time from 1 h over 5 h up to the standard of 24 h (Fig. 2a, c). It was observed that lower amount of PEGda concentration (2.5 %) compared to standard protocol (5 %) leads to bigger pore sizes as well as thinner pore walls (pictures not shown). These results could be confirmed by measuring the average pore size, which are decreased (Fig. 2d). Whereas total porosity is influenced in both cases and leads to a significantly decrease down to 10 %. Doubling the amount of PEGda to 10 % leads to an increase of elasticity from 40 to 65 kPa and halving it to 2.5 % decreases elasticity from 40 to 10 kPa (Fig. 2d).

Influence of scaffold properties on proliferation and spheroid formation of epithelial prostate cancer cell line LNCaP
As we could demonstrate, the lack of gelatin (approach 1) has no negative effect on the physical properties (Fig. 2b). Nevertheless, gelatin contains the RGD sequence, which is essential for cell adhesion. Interestingly the effect of this chemical attractants on cell growth is not as much as expected (Fig. 3a), but the absence of gelatin leads to a minimization of the spheroid size from 0.009 mm 2 (within the standard cryogel) to 0.004 mm 2 (Fig. 3b). However, spheroid shape looks similar-round and regular-independently of RGD presence (Fig. 3c).
After 3 weeks an obvious reduction in proliferation was obtained, which suggests proliferation stop or even dying of cells (Fig. 3a, approach 2, 1 h). The impact of higher elasticity is already visible with cryogels frozen for 5 h, which shows only small differences to proliferation rate of cryogels frozen for 24 h (Fig. 3a, approach 2, 5 h). A stiffer cryogel (Fig. 3, approach 3, 10 % PEGda) improves proliferation at day 21. To investigate, if our results are dependent on the cell type we measured additionally the proliferation rate of the prostate cancer cell line PC3 according to the influence of RGD presence and elasticity. The presence (standard) or absence of RGD (approach 1) has no significant influence on the proliferation rate of LNCaP as well as PC3 (Table 1). In contrast to that stiffer cryogels (approach 3, 10 % PEGda) increase growth of PC3 cells only. Whereas soft matrices (approach 3, 2.5 % PEGda) reduce cell growth in both cases.
Furthermore, it was observed that support of nutrient transport by suitable porosity and pore size combined with stable scaffold given by elasticity, leads not only to the highest cell proliferation, but also to well-and round-shaped spheroids with a volume of nearly 0.01 mm 3 (Fig. 3b, c, standard). Spheroid shape is independent of chemical attractants like RGD sequences, which should improve cell adhesion to the matrix, however decreasing spheroid size to 0.004 mm 2 (Fig. 3b, c, approach1).
The principle low growth rate and even decreased proliferation of cells within cryogels, which was freezed for only 1 h (Fig. 3a, approach 2), becomes coherent by analyzing spheroid volume and shape. In all spheroids is a necrotic core visible (Fig. 3c, approach 2). Interestingly the spheroid size of 0.001 mm 3 is comparable with the volume of spheroids grown in cryogels of approach 2, which was freezed for 5 h (Fig. 3b). Nevertheless, proliferation rate was positively affected by increased porosity caused by longer freezing time of 5 h (Fig. 3a, approach2). For cell proliferation the differences in elasticity seems to have no influence within the first 14 days of LNCaP cell culture (Table 1), whereas a long-term cultivation needs mechanical strength and stiffer material to increase cell growth (Fig. 3a). Interestingly spheroid size decrease in case of higher stiffness (Fig. 3b, approach 3, 10 % PEGda) more than with low elasticity (Fig. 3b, approach 3, 2.5 % PEGda) compared to spheroid size of standard cryogels.

LNCaP
In a scaffold without gelatin, like it was given in approach 1, no RGD sequence is present. We could observe that this fact leads to a cytoplasmatic localization of AR, although hormone is still available (Fig. 4, approach 1). Furthermore, in our work we could investigate Fig. 2 Physical characteristics (total porosity, average pore size and elasticity) of a standard, b approach 1 (without gelatin), c approach 2 (changes freezing time) and d approach 3 (changed PEGda concentration cryogels. Porosity was measured by mercury intrusion porosimetry and elasticity by compressive strength test. The experiments were done in triplicate. Error bars represent standard faults by applying approach 2 that less porosity and pore size leads also to an AR location in cytoplasm (Fig. 4, approach 2, 1 h). It seems to be that after 5 h freezing time and thus doubling the porosity as well as the average pore size AR is localized frequently in the nucleus (Fig. 4, approach 2, 5 h). A similar behavior could be observed by changing elasticity with applying approach 3. Decreasing elasticity to around 10 kPa leads to an AR localization in cytoplasm, whereas AR is represented in nucleus by increasing stiffness of matrix to nearly 60 kPa (Fig. 4, approach 3).

Discussion
After generating a 'standard' cryogel and 3 variations of that according to chemical composition, pore network as well as elasticity the main physical characteristics were analyzed. The absence of gelatin (approach 1) in the material composition affects obviously none morphology nor elasticity. For cryogelation process the formation of ice crystals is the most important parameter as a template for generating the macroporous network, which can be tuned by freezing temperature and time. It is known that a freezing temperature of −10 to −21 °C leads to an optimal interconnected ice crystal system (Svec 2010). Therefore, we kept the freezing temperature stable at −21 °C and usually the optimal proposed period of 24 h. To investigate the principle influence of freezing time we focused on that in approach 2 and reduced the freezing time in two steps to 1 and 5 h, what influences mainly porosity and pore size. Whereas the cross linker concentration (approach 3) the mechanical strength and elasticity. The higher the amount of cross linker, the stiffer is the material. The fact that porosity decrease in correlation to stiffer and denser material with small pore sizes and thick pore walls (10 % PEGda), but also in correlation with very spongy and elastic material, is reasoned by collapsed pores under pressure and allows the fluid (i.e., mercury) during the measurement to be pressed through the material and enlarge by this way the pores (2.5 % PEGda). Hence, mechanical strength influences indirectly the pore network. The cryogel formation recommends that the cryogelation process depends upon the temperature of freezing and the rate of crosslinking. An increasing of initial monomer/polymer as well as cross linker concentration leads to a faster crosslinking rate at the same temperature (−21 °C). The gelation process in the polymer solution starts before the porogens (ice crystals) are formed (Kathuria et al. 2009). The faster crosslinking rate implicates formed gels with less porosity, less mechanical strength and brittle nature. The current study reveals the possibility to manipulate the cryogel properties with single parameter of the synthesis process including material composition, freezing time and amount of cross linker. In order to investigate the effect of chemical and physical scaffold characteristics on 3D cell culturing to design an appropriate prostate model, we initially cultivated the epithelial prostate cancer cell line LNCaP within the varied cryogel matrices up to 21 days and analyzed growth, spheroid formation as well as androgen receptor (AR) localization.
Proliferation analysis showed that the lack of chemical attractants such as RGD sequence can be compensated by a suitable porosity and pore size. And vice versa, the combination of too less porosity, small average pore size and low elasticity cannot be compensated, even not by presence of RGD. Thus, incorporation of chemical attractants such as RGD might be important for cell attachment on the scaffold especially for the first days, but is ineffective and not necessary anymore, respectively, for long-term 3D culturing regarding growth rate.
We suggest therefore, that long-term culture of cells depends more on sufficient elasticity, which stands for tissue-like behavior as well as for mechanical strength and sufficient nutrient supply given by stabilization of the pore network. Nevertheless, the higher elasticity (approach 3, 10 % PEGda) did not increase cell proliferation compared to the standard cryogel. But this result could be reasoned by applying LNCaP cell line, which is relatively independent to effects of stiffness (Tilghman et al. 2010). We could confirm this hypothesis by applying another prostate cancer cell line (PC3), which is highly metastatic and derived from bone metastasis (Kaighn et al. 1979). Stiffer cryogels reduplicate the growth rate of PC3. We suggest that higher E modulus of 3D matrices stimulates growth of cells which are of metastatic origin and tend to metastasize after a certain time in culture, respectively. Furthermore, RGD presence or absence showed no effect on cell growth also in this case.
H&E staining enabled to analyze the formation and size of the tumor spheroids. The smaller the average pore size, the smaller the spheroid. Increasing cross linker concentration to design stiffer cryogels (approach 3, 10 % PEGda) affects porosity and pore size negatively by forming thicker pore walls and a denser matrix in general. Interestingly same effects could be obtained by decreasing elasticity (approach 3, 2.5 % PEGda), which is in this case due to the collapse of pores given by mechanical weakness. Therefore, it is possible to minimize spheroid size and maintaining high cell proliferation by doubling the stiffness of the surrounding scaffold.
In our study we checked the androgen receptor (AR) localization by immune fluorescent staining of LNCaP cells cultured for 21 days within the various cryogel types. Androgens play a critical role in the development and progression of PCa by controlling androgen-regulated genes (ARGs) mediated via AR, a transcription factor. Upon ligand binding the cytosolic AR is translocated into the nucleus, where it binds to the promotors of ARGs. It is well-known that AR signaling and androgen responsiveness of LNCaPs appear to be sensitive to serial passaging and different culture conditions. Indeed, we could conclude from our work that AR localization depends on RGD presence, porosity and elasticity. The fact that RGD and the interaction between tumor cells and ECM, respectively, affect expression of ARGs was already investigated by other workers (Robbins et al. 1996;Sieh et al. 2012). But as far as we know this is the first hind that porosity and elasticity of surrounding environment have a direct and independent influence on cell behavior over AR localization. It seems to be that certain porosity as well as a certain elasticity range leads to remaining of AR in the cytoplasm although hormones are present in the standard medium. Such findings confirm the strong need for a reliable model system for anti-AR drug screening, which mimic the in vivo situation as good as possible and avoid influences by the artificial surrounding as well.

Conclusion
For designing a well-defined 3D cell culture system to study prostate cancer it is mandatory to know about the crosstalk between matrix and cells. The absence of chemical attractants (i.e., RGD) does not influence physical parameters and interestingly does not improve cell proliferation, but reduces spheroid size. The proliferation rate depends strongly on physical scaffold properties and showed a stepwise increase of cell number by improving the pore network. The effect of less elasticity on cell proliferation is comparable with those results obtained in scaffolds with an insufficient pore network. The most outstanding effect we could examine was the influence of chemical and physical properties on the localization of the androgen receptor. It is expected that AR is localized in the nucleus to activate ARGs in the presence of hormones like it is in the case of applying standard medium. But storage in the cytoplasm could be observed in scaffolds without RGD, with a low porosity and a low elasticity. This fact is not related with spheroid size or proliferation rate.
Thus, this study is a step towards a tailor-made scaffold, which provided precise and well-defined environment focused on the so important rigidity and density of matrix for prostate cancer cells. Furthermore, only small variations in synthesis already enable to guide cells dependent on research questions which shall be answered with this system. Therefore, the suggested 3D cryogel scaffold could be the basis for developing a tunable cell culture platform for cancer research and screening for anticancer drugs, especially anti-AR drugs.