Abstract. – OBJECTIVE: In recent years, thefield of cancer immunotherapy has become a re-search hotspot and is currently faced with nu-merous challenges. The objective of this studywas to assess the success of cbl-b gene silenc-ing in splenic T lymphocytes as an immunestrategy to target the murine prostate cancerRM-1 cells in vitro and solid tumors in vivo.

MATERIALS AND METHODS: For this pur-pose, cbl-b gene-specific siRNA was designed,synthesized, and was transfected into mousesplenic T lymphocytes, followed by assessmentof T cell activation, TH1 cytokine production,and in vitro cytotoxicity against RM-1 cell tar-gets. For in vivo cytotoxicity studies, first theRM-1 tumor model was established in immunecompetent mice that were later tumor-injectedwith splenic T lymphocytes transfected withspecific shRNA for cbl-b gene silencing.

RESULTS: The data show that the cbl-b genesilencing in T lymphocytes resulted in an en-hanced surface expression of CD69 activationmarker, elevated production of interleukin (IL)-2and interferon (IFN)-γγ, and their increased cyto-toxicity as effectors against RM-1 prostate can-cer cells. The tumor injection with cbl-b shRNA-transfected T lymphocytes also resulted in sig-nificant reduction of the tumor size as com-pared with controls.

CONCLUSIONS: cbl-b gene silencing strate-gy enhanced the immune function of T lympho-cytes, increased their cytotoxic potentialagainst RM-1 prostate cancer cells, as well ascaused significant suppression of the tumorgrowth in immune competent mice.

Corresponding Authors: Cong-Hui Han, MD; e-mail: bujiniao2008@qq.com;S. Li, MD; e-mail: 13583181060@163.com 3819

Key Wordscbl-b, Prostate cancer, siRNA, shRNA, T lymphocytes,

Tumor immunity.

Introduction

Prostate cancer is the most common malignan-cy in elderly males. In China, the incidence ofthis disease has been less than that in the Westerncountries. However, there is an increasing trendobserved in the incidence of the disease in thiscountry. In China, 80.6% patients with definitivediagnosis of prostate cancer were identified to beat the advanced and late stages of this disease,most of whom were radically unresectable1. En-docrine therapy and adjuvant radiotherapy re-main to be the mainstream of current interven-tions. However, many patients may suffer fromandrogen-independent prostate cancer which re-mains poorly responsive to endocrine therapyand adjuvant radiotherapy, resulting in poor over-all survival (OS) rates. As the 4th interventionaloption, tumor immunotherapy based on tumor bi-ological treatment has received increasing atten-tion in recent years. Cumulative evidence haspointed to the development of immune toleranceregarding tumor-specific lymphocytes in localmicroenvironment, resulting in failure of tumorimmunity2-6. Therefore, mono-immunotherapy

European Review for Medical and Pharmacological Sciences

Cbl-b gene silencing in splenic T lymphocytes as a therapeutic strategy to target the prostate cancerRM-1 cell tumors in immune competent mice

Z.-D. SHI1, X.-F. LI3,8, L. HAO1, Y. ZHAO1, Y.-X. WANG5, B.-Z. DONG1, W.-H. CHEN2, Z.-G. ZHANG1, Y.-M. WANG2, Q. FU6, C.-H. HAN1,4, S. LI7

1Department of Urology, The Affiliated School of Clinical Medicine of Xuzhou Medical College,Xuzhou Central Hospital, Xuzhou, China2Department of Urology, Shanghai East Hospital, Shanghai, China3Shandong University, Shandong, China4Kunshan City Ruike Drug Development Company, Kunshan, China5Department of Nephrology of Xiamen NO.2 Hospital, Xiamen, China6Department of Urology, Shandong Provincial Hospital, Shandong University, Jinan, China7Department of Urology, Shandong Province Hospital of Traditional Chinese medicine, �AffiliatedHospital of University of Traditional Chinese medicine (TCM), Jinan, China8Department of Reproductive Medicine, Binzhou Medical University Hospital, Binzhou, China

Z.-D. Shi, X.-F. Li, L. Hao, Y. Zhao and Y.-X. Wang contributed equally to this work and should be considered as co-first authors

2014; 18: 3819-3830

3820

tended at increasing the tumor-specific antigenpresenting cells, such as administrating in vitroexpanded tumor-infiltrating lymphocytes (TILs),may not be successful. The development of tu-mor-induced immune tolerance is a multi-factori-al process that induces lymphocyte suppression,resulting in failure of T cells to produce an effec-tive immune attack against tumor cells.Importantly, correlations between prostate

cancer progression and paracrine TGF-β signal-ing as well as between tumor invasion/metasta-sis and expression profile of inhibitory CD28ligand B7-H3/B7x have been reported3-7. Thekey to the success of antitumor immunity andimmunotherapy is how to effectively activatethis population of tumor-specific immune cellsor reverse immune tolerance or immune incom-petent state. In this regard, E3 ubiquitin ligaseCasitas B-lineage Lymphoma proto-oncogene B(cbl-b) gene silencing strategy may help over-come the afore-mentioned challenges. Cbl-b is anegative regulatory molecule of T cells and itplays a vital role in T cell activation7-9; cbl-bgene defect could not only directly activate cy-totoxic T cells (CTLs) in the absence of co-stimulatory ligands9-13, but also reduced the sen-sitivity of T cells to suppressive cytokines suchas TGF-β and suppressive T regulatory cells(Tregs)14. Cbl-b gene deletion in mouse modelstudies was associated with decrease of tumorincidence and regression of existing tumors14,15.Cbl-b, a member of cbl family, was first isolatedfrom human breast cancer cells16. The cbl fami-ly contains negatively regulating signal mole-cules that are widely distributed inside the body.Cbl-b gene is located on chromosome 3 andcontains 19 exons; cbl-b protein has molecularweight of ~108kDa and acts as a signal mole-cule of various membrane receptors. It has dis-tinct structural domains, including a highly con-servative N-terminal, which mediates the bind-ing process of cbl-b protein with diverse intra-cellular molecules. Cbl-b exerts its role as aconjunction or scaffold molecule during theprocess of cell activation. In these domains,RING domains are dominant and contain E3ubiquitin chains which are associated with ubiq-uitin ligase activity of cbl-b for target proteindegradation and negative regulation of intracel-lular signal transduction17. This negative regula-tion is vital to maintain an appropriate immuneresponse and cbl-b suppression may induce au-toimmune disease. Cbl-b deletion was associat-ed with thymic hyperplasia, upregulation of

CD3 marker on T cells, increased Zap70 activi-ties, and enhanced T cell proliferation in re-sponse to CD3 antibody stimulation18.Herein, based on an immunocompetent tumor-

bearing mouse model of prostate cancer, cbl-b-specific siRNA was transfected into T lympho-cytes to silence cb1-b expression in order to: (i)reverse the immune tolerance of tumor-specificlymphocytes; (ii) improve the immune vitality oftumor-specific lymphocytes; and (iii) evaluatesuppressive effects on prostate cancer and itsclinical application potential. Moreover, themechanisms of tumor immune escape and antitu-mor immunity were also investigated.

Materials and Methods

MiceThirty adult male (6 weeks old), healthy

C57BL/6 mice were procured from the ChineseAcademy of Science. The animals were housedin individual cages under 12h/12h dark/light cy-cle and were offered feed and water ad libitum.The study protocol was approved by the institu-tional Committee on Animal Research and Ethics(CARE) and all animal experiments performedconformed to the national and internationalguidelines for animal use in research.

Collection of splenic lymphocytes and cultureA healthy mouse was sacrificed by cervical

dislocation and was immersed in 75% alcohol for2-3 min for disinfection. After transferring to anultra clean bench, the abdominal cavity of mousewas cut open using small pair of scissors andspleen was removed. After excising the outermembrane and connective tissues, the residualmass was placed in a sterile Petri dish containingsmall amount of physiological saline. The spleenwas scratched with the frosted face of two slides,and washed with saline. Harvested fluid was fil-tered and 4 ml of filtrate was gently overplayedon 8ml of lymphocyte separation medium andthe tube was centrifuged in swinging bucket rotorat a speed of 1200 ×g for 20 min. Splenic lym-phocytes visible as a buffy coat at the interfacewere gently collected, mixed with normal salinefor washing, centrifuged at 800 ×g for 5 min andthe supernatant was discarded. After a total of 3washings, lymphocyte pellet was dislodged byfinger tapping, mixed with lymphocyte mediumand cells were counted.

Z.-D. Shi, X.-F. Li,�L. Hao, Y. Zhao, Y.-X. Wang, B.-Z. Dong, W.-H. Chen, et al.

Mouse splenic lymphocytes were cultured(2×106 cells/ml) using lymphocyte medium con-taining ConA (0.05 µg/ml) in a 6-well plate (2 mlper well) and incubated at 37°C in a humidifiedCO2 (5%) incubator for 3 days. Fresh culture medi-um (1 ml) was replenished after 2 days by replacingthe same volume from each well. In the event ofprolonged incubation, culture medium was replacedas before after every 2-3 days as required.

siRNA transfectionFour cbl-b specific siRNAs were designed and

synthesized by Shanghai Jikai Gene Co. Ltd.(China) and the sequences were as follows: siR-NA-1: 5'-UCC CAA GCU UCA GUU GAA Att-3'; siRNA-2: 5'-CCC UGA UUU AAC CGGAUU Att-3'; siRNA-3: 5'-CCA UCU CAU UGCCAU AAU Gtt-3'; siRNA-4: 5'-UGA GAU GCCCUG AUA UUA Att-3'. Mouse splenic lympho-cytes cultured in a 6-well plate for 3 days werewashed twice, resuspended to have a single cellsuspension in Opti-MEM by repeated pipetting,and cells were seeded (100 µl/well) in a 96-wellplate at a concentration of 1×105 cells/ml. Lipo-fectamineTM2000 liposomes (0.5 µl) and cbl-bsiRNA (1.5 µl) were added to 5 µl of Opti-MEM,gently mixed together and incubated at roomtemperature for 20 min. The transfection mixturewas added drop wise to designated well, dis-persed by gentle tapping before adding anti-CD3(20 µl), and under ambient conditions for 6h, af-ter which, 20 µl of heat-inactivated fetal bovineserum (FBS) was added and incubated at 37°C ina humidified CO2 (5%) incubator for another48h. The transfection efficiency was 89.42% (da-ta not shown).

Flow cytometryAfter 48h incubation, cbl-b-shRNA transfected

cells were harvested by centrifugation at 1200 ×gfor 5 min and suspended in 1ml of lymphocyteculture medium. Cells were counted, washedthrice in phosphate buffered saline (PBS) andsuspended (1×106) in 100 µl saline and incubatedwith 1 µl FITC-conjugated anti-CD69 mouse an-tibody at room temperature for 30 min in thedark. Cells were washed thrice as before and re-suspended in 500 µl of PBS. Cells were analyzedby flow cytometry (Becton and Dickinson,Franklin Lakes, NJ, USA) and data analyzed byCellQuest software. A total of 1×105 cells wereanalyzed for each sample and results were ex-pressed as percentages of fluorescent positivecells.

Western BlotSamples (each containing 100 µg protein)

were mixed with 2× sample buffer, boiled in100°C water bath and loaded in gel for sodiumdodecyl sulfate polyacrylamide gel electrophore-sis (SDS-PAGE). The gel was run first at 100 Vfor 10 min and then at 80 V for 2-3h. Proteinswere electro-transferred to PVDF membrane at80V for 1.75h. The membranes were blockedwith 1%BSA in tris buffered saline tween (TB-ST) at room temperature with shaking for 1h.The samples were incubated with primary anti-body (1:1000 diluted) at 4°C overnight withshaking. Samples were washed twice with TBST(5 min each time), followed by TBS wash twice(5min each time), then incubated with secondaryantibody (1:1500 diluted) at room temperaturefor 45 min with shaking. Samples were washedwith TBS for three times. The images were cap-tured and band intensity of target proteins wasanalyzed using GelAnalyse imaging analysissoftware. The results were expressed as band in-tensity of target protein divided by that of inter-nal reference.

RM-1 prostate cancer cell cultureRM-1 tumor cells, originally derived from

C57BL/6 mice, were rapidly defrosted at 37°C in awater bath, washed twice with physiological salinesolution and cultured (103 cells/ml) using RPMI1640 medium supplemented with 10% (v/v) FBS,100 U/ml penicillin, 100 µg/ml streptomycin in acell culture flask and incubated at 37°C in a hu-midified CO2 (5%) incubator for 2 days. Tumorcells were regularly passaged by trypsinization(0.05% v/v trypsin, 0.53 mM EDTA) during thelogarithmic growth phase as required.

T lymphocyte proliferation assayAfter 48h incubation, cbl-b siRNA-transfected

lymphocytes were incubated with cell counting kit(CCK)-8 solution (10 µl/well, at a volume ratio of1:10) at 37°C in a humidified (5% CO2) incubatorfor 1-4h. The absorbance values were determinedusing microplate reader at 450 nm and 630 nm.Triplicate readings were taken and the results wereexpressed as the average of three readings.

T lymphocyte in vitro cytotoxicity assayRM-1 prostate cancer cells (designated as target

cells) were centrifuged at 800 ×g for 5 min aftertrypsin digestion, followed by addition of lympho-cyte preparation medium (1 ml). Cell suspension(0.1 ml) was mixed with 0.4% Trypan blue for

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Cbl-b gene silencing in splenic T lymphocytes as a therapeutic strategy to target the prostate cancer

3822

counting, cell concentration was adjusted to1×106/ml, and 10 µl of cell suspension was addedto each well of a 96-well plate. T lymphocyteswith or without cbl-b gene transfection (designat-ed as effector cells) were inoculated into triplicatewells at the effect: target (E:T) ratios of 40:1, 20:1,and 10:1 and the volume was adjusted to 100 µl ineach well. Effector-target coculture (triplicate)plates were then incubated at 37°C in 5% CO2 hu-midified incubator for 24h, 48h, or 72h, followedby CCK-8 assays (Shanghai Bogu Biological Co.,Shanghai, China). The CCK-8 assay allows forsensitive colorimetric determination of the cell vi-ability in cytotoxic or cell proliferation assays. Inthis assays, Dojindo’s highly water soluble tetra-zolium (WST)-8 salt is reduced by dehydrogenaseactivities in live cells to generate a yellow colorformazan dye which is soluble in tissue culturemedia. Thus, the amount of formazan dye generat-ed by cell dehydrogenases is directly proportionalto the number of live cells present. The assay isbased on 3 simple steps: (1) addition of CCK-8solution; (2) incubation; and (3) optical density(OD) measurement at 450nm, which were per-formed following the manufacturer’s instructions.The rate of killing by T lymphocytes was deter-mined as follows:

T lymphocyte killing rate (%) = [1-(ODE+T – ODE/ODT)] × 100

Where, ODE+T: OD values of ‘effector plus tar-get’ cells; ODE: OD values of effector cells at cor-responding concentrations; and ODT: OD valuesof target cells at corresponding concentrations.

Inoculation of RM-1 cells into C57BL/6 miceAfter trypsinization of RM-1 prostate cancer

cells in logarithmic growth phase, the cells werewashed twice with physiological saline solutionand resuspended in culture medium. C57BL/6mice were transferred to ultra-clean bench, fur nearthe right armpit was trimmed off using curved scis-sors, and skin was sterilized with 75% alcohol.RM-1 tumor cell concentration was adjusted to2×107/ml and 0.1 ml of cell suspension was mixedwith biological glue on ice at a ratio of 1:1 and thepreparation was injected (0.2 ml) into the rightarmpit and the tumor growth profile was examinedperiodically. The diameters of tumors were mea-sured using a Vernier caliper and documented onceper three days after initiation of tumor formation.The tumor growth curve was also plotted.

T lymphocyte inoculation into C57BL/6 miceT lymphocytes transfected with plasmids con-

taining cbl-b shRNA were homogenized withculture medium by pipetting to render a cell con-centration of 1×107/ml. Then, lymphocyte sus-pension (0.5 ml) was administrated subcuta-neously into the tumor site using a 1 ml syringeas the tumor size attained 0.2 cm in diameter.

Statistical AnalysisThe data obtained were analyzed by multi-fac-

tor analysis of variance (ANOVA) usingSPSS14.0 statistical software (SPSS Inc., Chica-go, IL, USA). A p-value of < 0.05 was consid-ered to be statistically significant.

Results

Cbl-b protein expression after siRNA transfectionTotal proteins were extracted following the

manufacturer’s instructions using total proteinextraction kit (Shanghai Bogu Biological Co.Shanghai, China) from four batches of cbl-b siR-NA-transfected T lymphocytes as well as fromcontrol group and blank group (untransfected)lymphocytes. Protein concentrations were deter-mined by using Folin-phenol kit, expression ofcbl-b protein was determined by Western blotand results were analyzed using ShineTech Gel-Analyse image processing software. Cbl-b pro-tein suppression rates in 4 siRNA-transfected Tlymphocytes batches (Lanes 1 through 4; Figure1) were found to 62%, 13%, 8%, and 85%, re-spectively, while negative control (mock-trans-fected) and blank control (untransfected) lym-phocytes had comparable cbl-b protein expres-sion.

Surface expression of T cell activation marker CD69 after cbl-b gene silencingSurface expression of CD69 which is consid-

ered a useful marker for the early T lympho-cytes activation in mixed populations was alsodetermined by flow cytometry. The expressionof CD69 was significantly upregulated(6.37±0.40%) following siRNA-mediated sup-pression of cbl-b in T lymphocytes as comparedwith its expression in control (1.94±0.81%) andblank (1.85±0.20%) groups. The representativedata from three independent experiments areshown (Table I; Figure 2).

Z.-D. Shi, X.-F. Li,�L. Hao, Y. Zhao, Y.-X. Wang, B.-Z. Dong, W.-H. Chen, et al.

IL-2 and IFN-γ production byT lymphocytes after cbl-b gene silencingThe expression of IL-2, a Th1 cytokine that is

centrally involved in the growth and proliferationof T lymphocytes as well as their differentiationinto ‘effector’ T cells, was determined in T cellculture supernatants using commercial ELISA kit(Shanghai Bogu Biological Co., Shanghai, China)and following the manufacturer’s instructions. IL-2 secretion by T lymphocytes was significantlyupregulated (p < 0.01) after siRNA-mediated cbl-b gene suppression in experimental group(852.78±38.17 pg/ml) as compared with its ex-pression in control (629.46±37.00 pg/ml) andblank (593.83±15.98 pg/ml) groups (Table II).Also, the expression of interferon (IFN)-γ, a

cytokine that is critical for innate and adaptiveimmunity against viral/intracellular pathogens ortumors and is secreted by both T lymphocytes andnatural killer (NK) cells, was determined in T cellculture supernatants using commercial ELISA kit(Shanghai Bogu Biological Co., Shanghai, China)and following the manufacturer’s instructions.IFN-γ secretion by T lymphocytes was signifi-

cantly upregulated (p = 0.002) after siRNA-medi-ated cbl-b gene suppression in experimentalgroup (208.19±7.92 pg/ml) as compared with itsexpression in control (167.08±10.97 pg/ml) andblank (163.76±7.35 pg/ml) groups. The represen-tative data from three independent experimentsare shown in Table II.

T lymphocyte proliferative responsesfollowing cbl-b gene silencingT lymphocyte proliferative responses were also

assessed following siRNA-mediated suppressionof cbl-b gene by using CCK-8 kit as describedearlier in materials and methods. Consistent withincreased IL-2 expression, T lymphocyte prolifer-

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Cbl-b gene silencing in splenic T lymphocytes as a therapeutic strategy to target the prostate cancer

Figure 1. Suppression of the cbl-b protein expression. Suppression of cbl-b protein expression in the mouse splenic T lym-phocytes following transfection with 4 different siRNAs was determined by Western blot, using β-actin housekeeping gene asinternal control. Lanes 1 through 6 represent siRNA-1, siRNA-2, siRNA-3, siRNA-4, blank (untransfected) and control (mock-transfected) lymphocytes, respectively. The cbl-b protein suppression induced by siRNA-1, siRNA-2, siRNA-3, and siRNA-4was 62%, 13%, 8%, and 85%, respectively. The representative blot from three independent experiments is shown.

Groups CD69 expression (%)

Experimental group 6.37±0.40(cbl-b siRNA-transfected)Control group 1.94±0.81(mock-transfected)Blank group (untransfected) 1.85±0.20

Table I. Surface expression (%) of activation marker CD69 on blood T lymphocytes after cbl-b gene silencing

Experimental vs. Control: q = 13.594 (p < 0.001); Experi-mental vs. Blank: q = 14.29 (p < 0.001); Control vs. Blank:q = 0.697 (p = 0.640).

Figure 2. Surface expression of CD69 T cell activationmarker. Surface expression (%) of CD69 activation markerwas determined by flow cytometry on blood T lymphocytesfollowing transfection with cbl-b siRNA-4. The representativehistograms from three independent experiments are shown.

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ation was also enhanced significantly (p < 0.05)in the experimental group (OD = 1.43±0.02) ascompared with control group (OD = 0.542±0.03).The representative data from three independentexperiments are shown in Table III.

T lymphocytes-meditated killingof RM-1 prostate tumor cellsT lymphocytes-mediated killing of RM-1

prostate tumor cells was determined using CCK-8 commercial kit as describe in materials andmethods. Higher cytotoxic activity by T lympho-cyte effectors was observed in the experimentalgroup as compared with control in effector-targetco-culture assays. The highest cytotoxicity(82.02%) was observed at an effector-to-targetratio of 40:1 at 24h (Figure 3; Table IV). Consis-tent with this finding, more extensive lymphocyt-ic infiltration together with plaques of floatingdead tumor cells were observed in cocultures at24h (supplementary Figure S-1).

T lymphocytes-mediated cytotoxicityin prostate cancer mouse modelC57BL/6 mice were inoculated subcutaneous-

ly in the armpit with RM-1 prostate cancer cellsto establish a mouse tumor model. The tumornodule started to appear at about 7 days post-in-oculation and, as shown in Figure 4, tumor ofnearly 2cm size was observed at 20 days. The tu-mor biopsy shows successful establishment ofsolid tumor with typical histopathological fea-tures of abundant cytoplasm, abnormal cellularmorphology, mitotic fat and skeletal muscle infil-tration, and angiogenic blood vessel growth (sup-plementary Figure S-2).Following intra-tumor injection with cbl-b

shRNA-transfected T lymphocytes, the tumorsize (diameter in cm) was measured every 3days, starting at the 9th day onward. The rate oftumor growth in experimental group (inoculatedwith shRNA-transfected T lymphocytes) was sig-nificantly lower than control (untransfected Tlymphocytes) and blank (un-inoculated) groups(Figure 5). As expected, the tumor regressionwas induced following intra-tumor injection withsplenic T lymphocytes transfected with cbl-bshRNA. The tumor at 4 weeks (Figure 6) was re-markably smaller in experimental mice (inoculat-ed with shRNA-transfected T lymphocytes) ascompared with control (inoculated with non-transfected T lymphocytes) and blank mice (un-inoculated); and at necropsy performed at 6weeks, the tumor diameter (cm) in experimentalmouse was significantly smaller than in controlmouse (Figure 7).

Discussion

Cbl-b, a member of Cbl protein family, is akey gene responsible for the immune functionregulation. As a negative regulatory gene, it playsa vital role in the activation of adjacent Tcells19,20. The strategy of cbl-b gene silencingcould not only directly stimulate CTLs in the ab-sence of co-stimulatory ligands to produce mas-sive IL-2 cytokine, but also reduce the sensitivityof T-cells to suppressive cytokines such as TGF-β, and subsequently kill tumor cells through ac-tive immunization which suggests a promisingpotential for their use in the field of tumor im-mune intervention. Accordingly, four cbl-b spe-cific siRNAs were designed and synthesized, andsubsequently transfected into mice splenic lym-phocytes using liposomes as vector. The transfec-

Z.-D. Shi, X.-F. Li,�L. Hao, Y. Zhao, Y.-X. Wang, B.-Z. Dong, W.-H. Chen, et al.

Groups IL-2 IFN-γ (pg/ml) (pg/ml)

Experimental 852.78±38.17 208.19±7.92group (cbl-b siRNA-transfected)Control group 629.46±37.00 167.08±10.97(mock-transfected)Blank group 593.83±15.98 163.76±7.35(untransfected)

Table II. IL-2 and IFN-γ concentrations in T cell supernatants after cbl-b gene silencing

Experimental vs. Control: q = 12.169 (p < 0.001); Experi-mental vs. Blank: q = 14.08 (p < 0.001); Control vs. Blank: q= 1.890 (p = 0.230).

Groups Optical Density (OD)

Experimental group 1.43±0.02(cbl-b siRNA-transfected)Control group 0.542±0.03(mock-transfected)Blank group (untransfected) 0.528±0.07

Table III. T lymphocyte proliferative response following cbl-b gene suppression

Experimental vs. Control: q = 119.571 (p < 0.001); Experimen-tal vs. Blank: q = 121.553 (p < 0.001); Control vs. Blank: q =1.982 (p = 0.211).

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Cbl-b gene silencing in splenic T lymphocytes as a therapeutic strategy to target the prostate cancer

Figure 3. In vitro cytotoxicity ofT lymphocytes against RM-1prostate cancer cells. T lympho-cytes-mediated killing (%) of RM-1tumor cells, as determined by usingCCK-8 commercial kit, is shown at(A) 24h; (B) 48h; and (C) 72h. Sig-nificantly higher cytotoxicity wasobserved by T lymphocyte aftersiRNA-mediated cbl-b gene silenc-ing as compared with control. Thehighest cytotoxicity of 82.02% wasobserved at an effector to target(E:T) ratio of 40:1 at 24h. The dataobtained from four independent ex-periments are shown.

Duration Cbl-b siRNA-transfected lymphocytes Untransfected lymphocytes

10:1 20:1 40:1 10:1 20:1 40:1

24h 38.36±1.82 49.27±1.66 82.03±1.82 28.76±1.83 38.67±1.75 57.94±2.3148h 36.68±1.76 49.03±0.57 73.17±2.31 27.74±2.11 37.89±1.47 56.60±1.4672h 28.08±2.53 45.51±1.24 68.31±1.97 21.33±1.78 30.47±0.69 53.78±1.58

Table IV. Cytotoxicity (%) of cbl-b siRNA-transfected and untransfected T lymphocytes against RM-1 prostate cancer cells.

Significant differences were observed between Cbl-b siRNA-transfected and untransfected cells with regard to time (24h, 48h,72h) and effector: target ratios (10:1, 20:1, 40:1) (All p-values

3826

tion efficiency was found to be 89.42% whichwas higher than that obtained through othertransfection methods used elsewhere. In order tofurther assess the siRNA-mediated suppressiveeffect on cbl-b gene expression, cbl-b protein ex-pression was determined by Western blot at 48hwhich indicated the variable degree of cbl-b geneexpression by 4 siRNAs used and siRNA-4 in-duced the highest gene expression at protein lev-el (85%) in mouse splenic T lymphocytes. Dif-ferent silencing effects are generated by siRNAstargeting different nucleotides of the same mR-NA transcribing into variable expression of cbl-bprotein. In this study, we found that siRNA-4 in-duced the maximum suppression in cbl-b geneexpression. Consistent with this finding, it wasreported earlier that the effective suppression ofgene expression could be observed by only fewsiRNAs21.

E3 ubiquitin ligase cbl-b is a key factor in-volved in immune regulation and cbl-b upregula-tion has been associated with development of pe-ripheral tolerance and anergy of T cells22. Thus,cbl-b is critical for establishing the threshold forT cell activation. Since cbl-b functions as a nega-tive regulator of T cell activation, we assessed thesecretion of two immune regulatory T cell cy-tokines (IL-2 and IFN-γ) in the supernatants ofmouse splenic T lymphocytes that were transfect-ed with cbl-b-specific siRNA. IL-2 is the majorfactor for T cell activation and proliferationwhich is foundation of priming the feedbackloop. IFN-γ is a type II interferon that has antivi-ral, antitumor and immunoregulatory properties.As the data show, both IL-2 and IFN-γ werefound significantly upregulated in T cell super-natants of experimental group as compared withthose of control or blank groups; thus, suggesting

Z.-D. Shi, X.-F. Li,�L. Hao, Y. Zhao, Y.-X. Wang, B.-Z. Dong, W.-H. Chen, et al.

Figure S-1. Lymphocytic infiltration of RM-1 prostate cancer cell cultures at 24h (magnification: 100×). Cbl-b gene silencingvia siRNA transfection induced T lymphocyte activation and extensive lymphocytic infiltration of RM-1 prostate cancer cellswas observed at 24h in experimental group (A) as compared with control group (B). Plaques of floating dead cells (arrow)were frequently visible in experimental cocultures.

Figure 4. Establishment of prostate cancermouse model. C57BL/6 mice were inoculat-ed subcutaneously in the right armpit withRM-1 prostate cancer cells, as described inMaterials and Methods, to establish a mousetumor model. The development of tumornodule was visible at about 7 days post-in-oculation and a solid tumor of ~2 cm size(arrow) was observed at 20 days.

a significant amplification of the lymphocyte ac-tivity following cbl-b gene silencing. Consistentwith our findings, Stromnes et al23 also reportedthat siRNA knockdown of Cbl-b in CD8+ effectorT cell clones enhanced the IL-2/IFN-γ produc-tion, restored T cell proliferative responses andincreased target avidity.To further investigate the cytotoxicity of cbl-

b-knockdown lymphocytes against tumor cells,T lymphocytes with or without cbl-b siRNAtransfection were cocultured with RM-1prostate cancer cells at E:T ratios of 40:1, 20:1and 10:1. The cbl-b gene knockdown lympho-cytes (i.e. siRNA transfected) showed signifi-

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Cbl-b gene silencing in splenic T lymphocytes as a therapeutic strategy to target the prostate cancer

Figure S-2. Tumor histopathology at 2 weeks. The tumorbiopsy performed at about 2 weeks shows the successful es-tablishment of solid tumor with typical histopathologicalfeatures including tumor cell infiltration, abundant cyto-plasm, abnormal cell morphology, clear nucleoli, mitotic fatand skeletal muscle infiltration, and angiogenic blood vesselgrowth.

Figure 5. Rate of tumor growth inC57BL/6 mice. Following intra-tumor injec-tion with cbl-b shRNA-transfected T lym-phocytes, the tumor size (diameter in cm)was measured every 3 days, starting at the9th day onward. The rate of tumor growth inexperimental group (inoculated withshRNA-transfected T lymphocytes) was sig-nificantly lower than control (untransfectedT lymphocytes) and blank (un-inoculated)groups.

cantly higher (p < 0.05) cytotoxicity against thetumor cells as compared with controls; and thehighest killing (85%) was observed at E:T ratioof 40:1 at 24h. This observation suggests thepossibility of amplifying cytotoxicity againstRM-1 prostate cancer cells through the cbl-bgene knockdown/ablation strategy in in vivomodel studies. Since target cell killing rates at48h and 72h mutually differed non-significantlyand were relatively less than killing observed at24h, we propose that the time-dependent char-acteristic of siRNA interference may explain thereduced toxicity over time. Furthermore, sincekilling rates differed significantly (p < 0.05) be-tween E:T ratios used, the selection of an appro-priate E:T ratio and assay time would be criticalto the successful clinical application of this in-terventional strategy for prostate cancer im-munotherapy. Since mice bear similarities tohumans in many aspects of genetics, pathology,and biology, they have been widely used as ani-mal model for cancer research24,25. Using stateof the art technology, desirable gene mutationswith regard to human disease can be designedand induced to generate the relevant murinemodels. Thus, mouse models can be employedas a powerful tool to integrate the fundamentaland clinical cancer research in immune preven-tion and therapy.In order to establish an immune competent

mouse tumor model, RM-1 prostate cancer cellsharvested from logarithmic culture phase wereadministered subcutaneously into the rightarmpit of 6-month old mice and the tumor de-velopment started at one week post-inoculation.Since the directly administered tumor cells in

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immune competent mice could undergo phago-cytosis by effector cells, co-administration withbiological glue allowed for tumor cell immobi-

lization at the site of inoculation, resulting insuccessful development of tumor. Thus, our im-mune-competent mouse model was differentfrom the immune-deficient models that werepreviously used in related studies and has thepotential benefits of economics, feasibility, andefficiency for further use in subsequent studies.Besides, siRNA-mediated cbl-b gene suppres-sion validated in short-duration (3-day) ex vivocytotoxicity assays was considered unsuitablefor the in vivo cytotoxicity; we, therefore, usedplasmids expressing more suitable and long-act-ing shRNA for cbl-b gene interference in mousemodel studies. The data show that 3 out of 5mice administered with cbl-b shRNA-transfect-ed lymphocytes exhibited reduced tumorgrowth, higher activation status of the peripher-al blood T lymphocytes, and enhanced IL-2/IFN-γ production, whereas other two micewere comparable with controls. The differencemight be due to variations in the individual im-munobiological and genetic factors involvedwhich needs to be further investigated. Thisstudy sets the stage for further investigations in-to prostate cancer immune intervention via thecbl-b gene silencing, however, one major cau-tion relates to possible development of allergicauto-antigenic responses over time leading toautoimmune disease. Notwithstanding that suchautoimmune diseases are often non-lethal, ac-tive preventive and therapeutic measures need tobe considered in order to minimize any adversereactions associated with gene immunotherapy.Cbl-b gene silencing may lead to autoimmunedisease which, as compared to prostate cancer,is non-lethal; however, further studies will berequired to investigate the possibilities in differ-ent animal models.

Conclusions

Our in vitro study data show that siRNA-me-diated cbl-b gene silencing induced T lympho-cyte activation, increased cytotoxicity and en-hanced production of immunoregulatory cy-tokines, such as IL-2 and IFN-γ. Complement tothese findings, mouse model study data showthat the tumor-bed inoculation with cbl-bshRNA-transfected splenic lymphocytes leads totumor regression as assessed by tumor size re-duction. Taken together, cbl-b gene silencingstrategy may be used as a useful tool for prostatecancer immunotherapy.

Z.-D. Shi, X.-F. Li,�L. Hao, Y. Zhao, Y.-X. Wang, B.-Z. Dong, W.-H. Chen, et al.

Figure 6. Tumors at 4 weeks in C57BL/6 mice. The tumorregression was induced following intra-tumor injection withsplenic T lymphocytes transfected with cbl-b shRNA. Thetumor was smaller in experimental (inoculated with shRNA-transfected T lymphocytes) mouse (A) as compared withcontrol (inoculated with non-transfected T lymphocytes)mouse (B) and blank (un-inoculated) mouse (C).

–––––––––––––––––––-––AcknowledgementsThis study was supported by funds from the Research De-partment of Central Laboratory Shanghai East Hospital,Shanghai, China. Supported by the National Natural ScienceFoundation of China (81272557) and Natural Science Foun-dation of Jiangsu Province (BK2012647).

–––––––––––––––––––––Conflict of InterestThe Authors declare that there are no conflicts of interest.

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Figure 7. Tumor size at necropsy. The tumorsize (diameter in cm) at necropsy, performed at 6weeks post-inoculation, was significantly small-er in the experimental mouse (A) as comparedwith control mouse (B).

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