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Effects of Replacement of miR15 and miR16 expression in Chronic Lymphocytic Leukemia

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Figure 1

miR-15

miR-16

a mir15 mir16 b c d

Figure 1 Comparison between CLL carrying del13q and CLL without del13q (others). (a) Comparison of miR-15 and -16 expression in the 95 CLL cases with del13q miR-16 expression differences reached statistical significance (right panel, p<0.0001). (b) Del13q by FISH. Example of CLL sample with mono allelic 13q deletion, biallelic deletion or mixed cells. (c). Del13 q CLL cases were subgroup based on the heterogeneity of del13q as leukemic cells with prevalently biallelic del13q ; mono-biall del ; monoallelic del13q. Difference in miRNAs expression between CLL cases with bi-allelic or monoallelic are indicated (Mann-Whitney U test). (d) Mir15 and 16 expression validated in qRT-PCR

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Figure 2 Efficiency of miRNAs transfection (a) SmartFlareLive cell RNA detection (b) Example of Smartflare staining with mir15 (red) and miR16 (blu) in a normal Fish CLL(top); Biallelic del13q at time 0 (middle) and after transfection (bottom).(C) Mean of increase of miR15 and miR16 after transfection in 7 CLL cells. (d) Entry of miR15 and miR16 validated by qRT-PCR.

www.merckmillipore.com a

b c

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Figure 3 Inhibitor transfection in normal CLL(a) Expression of miR-15 and miR-16 following transfection with miR-15 (23.17 ±3) or miR-16 (16.5±2.8) or miR-CTR inhibitors (50.17 ±6.4 for miR-15; 38.17±4.2 for miR-16). Summary of tests on 6 CLL cases (CP0036, CR0203, PA0145, PM0608, CG0623, MA0342). (b) Smartflare staining with mir15 (red) and miR16 (blu). mir15 and miR16 at time 0 and after inhibitor transfection in a CLL without del13q © viable cells (%) are measured as Annexin-V/PI-double negative cells. The p-values were obtained by Wilcoxon test

Figure 3

miR-CTR miR-15 inhibitor miR-16 inhibitor

b a

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CLL cell viability in vitro following transfection with miRNA mimics (a) Determination of cell viability upon transfection with the indicated miRNA mimics. A representative time course experiment on cells from a CLL with biallelic del13q (MG0248) is shown. Viable cells (%) are measured as Annexin-V/PI-double negative cells. (b)Summary of viability determinations from 12 different biallelic del13q CLL cases after a 48-h culture following transfection of the miRNA mimics. p obtained with Wilcoxon test

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FIGURE 5 a
 % cell viability 0 10 20 30 40 50 60 70 80 90 100 LD0262 MA088 DT300 GM0041 CD300 VP0089 RR0108 MP0456 GD0051 MG0248 PA0254 GC0620

Figure 5 Effects of transfection with miR-15 and -16 in biallelic del (13)(q14) CLL cells. (a) Viability of purified CLL cells measured as Annexin V/PI negative cells, upon transfection with the indicated miRNA mimics after 48 hours of culture. Green box indicate CLL cases carrying TP53 mutations. (b) Western Blot (WB) analysis of BCL2, MCL1 and Survivin antiapoptotic proteins, or Cyclin D1 and D2 cell cycle proteins of a representative experiment in which purified CLL cells from case MP0456 were transfected with the indicated miRNA mimics and cultured for 72 hours. (c)

Summary of four experiments in different CLL cases (MP0456, GD0041,RD0296, PA0254), data are

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*

*

NS

G-CLL

NS

G-CTR

NGS-CTR NGS-CLL

Figure 6 MRI imaging and their relation with engraftment of the disease (a) two mice before and 24 hrs after iron uptake in NGS-CTR and NGS-CLL. (b-d)Matched spleen section by EE show presence of foci in CLL-mouse. IC evidenced absence of CD20+ (red) cells in NSG-CTR mouse and the presence of focal aggregates of CD20 positive cells in NSG-CLL spleen. In addition, Perls’ Prussian blue staining (used to detect USPIO nanoparticles) indicated that ferric iron particles were excluded from the focal lesions (Figure 6d right) whereas a random distribution of USPIO nanoparticles was observed in the spleens of NSG-CTR mice.

a

b

c

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spleen

αCD8

αCD4

αCD3

Figure 7 a CLL PATIENT Mouse spleen

αCD20

BcR TcR b

Figure 7 Example of IC in NGS mouse injected with CLL cells. (a) Paraffin spleen section stained with anti-CD20 and anti-CD4, anti-CD4 and anti-CD8 (b) BcR and TcR repertoire of human cells isolated from splenic mice and from PBMC of the corresponding patient.

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Figure 9

a b


% average reduction of CLL cells engraftment

Figure 9 CLL cell engraftment in NSG mice following in vitro transfection with miRNA mimics. Representative test on mice engrafted with CLL cells pre-treated in vitro with miRNA mimics (CLL CD0310),or miRNA-CTR. (a) MRI images 24 h after USPIO administration. The position of the spleen is indicated by the dotted red outline. ΔSNR% values also are indicated. The spleens with superior iron uptake and consequent lower ΔSNR% values, appear darker and less nodular. Conversely, spleens with lower iron uptake and higher ΔSNR% values are not so dark and show a nodular structure possibly related to the presence of follicles.(b) The CD20+ follicle-like structures are highlighted by red squares (magnification x40). The x200 magnification of a representative follicle for each panel is shown. c) Pooled flow-cytometry data obtained from 4 CLL cases with biallelic del(13)(q14) pre-treated with miRNA mimics in vitro before injection into mice (n=8 mice for each treatment group). The cells, harvested from mice at the end of tests, were stained and counted. Statistical comparisons were carried out using Wilcoxon-matched pair test. A P-value=0.0078 is indicated by **.

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a

b

c

Figure 10 CLL cell engraftment in NSG mice following in vitro transfection with miRNA inhibitors PM608

mice traded with miR-inhibitors or miR-CTR. Major iron uptake is related to a lower ΔSNR% values, and spleens appear darker and less nodular. In contrast, spleens with lower iron uptake and then higher ΔSNR% values are darkness and the nodular structure are possibly related to the presence of follicles.(b) α-CD20 Ab staining (red) of paraffin tissue embedded spleen samples following

injection of CLL cells pre-treated with the indicated miRNA. Pooled flow-cytometry data obtained from 2 mono allelic del13q CLL cases pre-treated with miRNA inhibitors in vitro before injection into mice (n=4 mice for each treatment group). The cells, harvested from mice at the end of tests, were stained and counted. Statistical comparisons were carried out using Wilcoxon text.

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198 119 841 5,6 Mir15 Mir16 Fold increase of mir15 and mir16 in MG0248 CLL cells Mouse1 Mouse2

Figure 11 Real time PCR analysis of CLL cells isolated from the spleen of mice treated with miRNA mimics. Analysis of the preliminary test performed in six NSG mice injected with MG0248 CLL cells and treated with a single potentially therapeutic dose of miR-15, miR-16 or miR-CTR complexed with

Invivofectamine. CLL cells (CD45+, CD19+ and CD5+) were purified by FACS sorting from splenic cell suspensions

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a

a b c

d

Figure 12 Effects of treatment with miR-15 or miR-16 mimics on the expansion of CLL clones in NSG mice. (a and b) IHC analysis of the spleen of a mouse a representative CLL case (MP0456).The typical CD20+ aggregates (a) surrounded by CD3+ T cells (b), that are evident in the spleens of the mice treated with miR-CTR, virtually disappear following treatment with miR-15 or miR-16 mimics. 40x magnification view of splenic sagittal sections. The inset indicates the same areas at a higher magnification c) Percent of CLL cells and T cells in mir15 and/or mir16 treated mice compared to miR-CTR. (d)

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FIGURE 13 0 50 100 150 200 250 300 350

FP0499 MONO GN0095 MONO RD0468 TRIS12 VS0624 NORM

FISH RM0626 DEL17p53 disf

IHC index

mirCTR miR-15 miR-16

Figure 13 Effect of mir15 and miR16 therapy in non biallelic del13q CLL following mir15 and miR16 treatment compared to miR-CTR. Bars represent mean of 2-3 mice for each group of treatment. Single data are reported in Table 2

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IHC is INDEX of Immunoistochemistry: delta SNR% is the percent of difference of the MRI signal intensity to noise ratio before and after USPIO control reagent injection

In blu are reported numbers of cases for which only one mouse could be tested by MRI

ID Treatment IHC index (mean

+SD) MRI delta SNR% (mean +SD)

Therapy started MRI deltaSNR% (mean +SD) Therapy completed MP0456 mir-CTR 59+4 +16+27 +36+25 mir15 9+3 +34+31 -47+3 mir16 4+1 +11+19 -66+11 PA0254 mir-CTR 67+9 +10 +33 mir15 33+6 +13 -59 mir16 31+2 -0.07 -48 DT300 mir-CTR 61+12 +44+14 +62+4 mir15 33+8 -81 -74 SG0232 mir-CTR 39+3 +3 +25 mir15 20+2 +1+2 -67+4 DA0346 mir-CTR 17+1 -0.81 +27 mir15 5+1 +19 -68 GM0041 mir-CTR 137+41 -1.5 +20 mir15 52+5 12 -34

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Patient Treatment IHC% (mean+ sd ) FP0499 miR-CTR 137+36,8 miR15 72+10,8 miR16 26+7,4 GN0095 miR-CTR 107+12,7 miR15 12+10 miR16 23+13,7 RD0468 miR-CTR 56+5,5 miR15 12+5,8 miR16 14+9,8 VS0624 miR-CTR 248+45,1 miR15 68+7,5 miR16 101+35,1 RM0626 miR-CTR 65+12,9 miR15 53+3,5 miR16 53+8,5

Table 2 :Inhibition of non-biallelic del(13q14) CLL cell growth in NSG mice by miR-15 or miR-16 mimic treatment

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