mirns regulate s Supplementary igures for MicroRNs Reprogram Normal ibroblasts into Cancer ssociated ibroblasts in Ovarian Cancer nirban K. Mitra, Marion Zillhardt, Youjia Hua, Payal iwari, ndrea E. Murmann, Marcus E. Peter & Ernst Lengyel Page of
mirns regulate s Direction of invasion djacent Matrigel DMEM + % S Microscope objective djacent ime (min) 6 8 4 6 8 4 6 8 4 4 6 8 [µm] old change speed p<. p<. djacent ig. S: Invasion of the patient-derived fibroblasts., Schematic of the dynamic in vitro invasion assay monitoring invasion of normal omental fibroblasts (s), adjacent s or cancer-associated fibroblasts (s) labeled with CMD (Cell racker Green). Invasion through matrigel was tracked by time lapse D confocal microscopy., Patient-derived s invade faster than s or adjacent s. Left, invasion of the indicated primary fibroblasts. Right, Quantification of the speed of invasion in the experiment shown on the left. Page of
mirns regulate s Direction of invasion OvCa cells djacent DMEM+% S Matrigel Microscope objective s + Hey8 Induced s + Hey8 [µ m] 4 6 8 4 6 8 ime (hrs) 4 old change speed p<. + +induced umor cells alone ime (min) 6 8 4 umor cells + + adjacent + 6 8 4 6 8 4 6 8 4 4 Hey8 4 6 Skovip 8 4 6 8 old change speed OVCR5 4 6 8 [µm] OvCa alone + + adjacent + ig. S: Characterization of s and induced s., op, schematic of an in vitro assay to follow invasiveness of ovarian cancer cells coincubated with either s, adjacent s, or s in real time using time lapse D confocal microscopy. ottom left, invasion of three OvCa cell lines co-incubated with the indicated patient-derived fibroblasts (s, as, or s). luorescently labeled cancer cells are in red, fluorescently labeled fibroblasts are green. Invasion through matrigel was tracked by time lapse D confocal microscopy. ottom right, quantification of the speed of invasion in assays shown on the left., Invasion of Hey8 cells co-incubated with s (top) or induced (i)s generated through seven days co-culture with Hey8 OvCa cells (bottom). he speed of invasion was quantified. Values represent mean ± s. d. Significance was calculated using Student s t-test. Page of
mirns regulate s Relative expression 5 4 ex vivo p=. p=.5 in vitro p=.5 p=.4 mir-4 mir- 5 mir-55 H&E a p=. p=. Induced mir-4 djacent umor H&E mir-55 djacent umor Page 4 of
mirns regulate s ig. S: Validation of mirn expression changes in s and induced s identified in the mirn array., RN was extracted from s isolated from the omental metastasis of patients with high grade serous OvCa (ex vivo) and matching adjacent s or from s and induced s derived from a two day co-culture of s with Hey8 cells (in vitro). he indicated mirns were quantified by quantitative real time PCR. Changes for corresponding pairs of fibroblasts for each individual patient are shown. Significance was determined using one-tailed paired t-test., In situ hybridization of human ovarian cancer omental metastasis and adjacent normal omentum for mir-4 (top) and mir-55 (bottom). Scale bar = µm. Stippled lines indicate the borders between areas of adipocytes () and fibroblasts () (rows and ) or tumor tissue () and fibroblasts () (rows and 4). Page 5 of
mirns regulate s old change.4...8.6.4...4...8.6.4.. LN (x) mir-4 mir- mir-55 pre-55 anti- anti-4 old change 4 8 6 4 8 6 4 8 6 4.4..8.6.4. Scr (x) LN mir-4 mir- mir-55 anti-55 pre- pre-4 ig. S4: Validation of mirn-transfection in s and s by quantitative real time PCR., Quantification of mirn expression using real-time PCR in s 48h after transfection with either ambled control oligonucleotides (ratio /LN :) or a combination of mirn inhibitors for mir- and 4 and pre-mir-55., Quantification of mirn expression using real-time PCR in s 48h after transfection with either ambled control oligonucleotides (ratio /LN :) or a combination of mirn inhibitor for mir-55 and pre-mir-4 and. Page 6 of
mirns regulate s C Migration Invasion Colony formation # of cells/field # of cells/field 8 6 4 Velocity (µm/h) D E 8 6 4 p<. LN anti- anti- 4 4 anti- LN 55 5 5 +Hey8 p=. 4 LN + Hey8 p=. p=. p=. anti- 55 # of colonies (fold change) 5 4 4 Hey8 Hey8 +Hey8 p<. p=.6 4 LN + Hey8 Migration Invasion Colony formation p=. p=.5 p<. 55 Velocity (µm/h) +Hey8 LN anti- anti- 4 + Hey8 55 # of colonies (fold change) +Hey8 LN anti- anti- 4 + Hey8 anti- 55 p=. 55 ig. S5. ltering individual mirn expression contributes to functional effects of mir-s. -C, Reprogramming of s to s. Overexpression of mir-4 (4), mirn- (), or inhibition of mir-55 (LN anti-mir-55) in s inhibits migration (), their ability to enhance Hey8 cell invasion (), and anchorage independent growth tested by colony formation (C). Negative controls () are indicated. D-, Reprogramming of s to s. Inhibition of mir-4 or mir-, or overexpression of mir-55 (55) in increases their migration (D), their ability to enhance Hey8 cell invasion (E), and anchorage independent growth (). Values represent mean ± s.d. from experiments performed at least three times. Significance was determined using one tailed t-test. Page 7 of
mirns regulate s mir- GUCGGUCGUGCGG 5 mir-4 CGGCGCCGGCG 5 ig. S6: he mir-4 seed match is the most highly conserved region in the CCL5 -UR of the human, mouse, and rat gene. lignment of the -URs of human, mouse, and rat CCL5. Identical nucleotide positions are marked by a red column. he positions of the seed matches of mir-4 and mir- are labeled. he -UR of human CCL5 was truncated. lignment of the -URs revealed that the region of highest identity ( nucleotides long) contains the seed match for mir-4. Page 8 of
mirns regulate s () IgG () anti-ccl5 mir- IgG mir- anti-ccl5 C () IgG () αccl5 mir- IgG mir- αccl5 % of Ki-67 positive cells 8 6 4 p<.5 () IgG p<.5 mir- () IgG αccl5 mir- αccl5 IgG anti-ccl5 IgG anti-ccl5 D IgG IgG αccl5 αccl5 % of Ki-67 positive cells 5 5 IgG p<.5 IgG p<.5 αccl5 αccl5 ig. S7: Orthotropic mouse xenografts., Representative bioluminescence images of mice (day 5) from igure 4 and, from igure 4. Color scale is in radiance (p/sec/cm /sr). he entire tumor for each mouse was quantified. C, Representative Ki-67staining of tumors from ig 4 (left), and quantification of Ki-67 staining in tumors from 4 mice/group (right). D, Representative Ki-67 staining of tumors from ig 4 (left), and quantification of Ki-67 staining in tumors from 4 mice/group (right). he significance was determined using one tailed t-test. Page 9 of
mirns regulate s CCR GPDH CCR 96 bp GPDH 96 bp HP- Hey8 SKOVip HP- Hey8 SKOVip Relative Expression 6 4 -vect -CCL5 ig. S8: Expression of CCR and CCL5., Expression of CCR in Hey8 and SKOVip ovarian cancer cells. otal RN from Hey8, Skovip and differentiated HP- (positive control) cells was converted into cdn followed by PCR for CCR or GPDH. he amplified products were resolved on a % agarose gel and stained with ethidium bromide. Representative image of two independent experiments is shown., Quantitative R-PCR for human CCL5 in s transfected with either human CCL5 vector or with vector control. Error bars represent triplicate experiments. Page of
mirns regulate s Normal fibroblasts () djacent Vimentin α-sm Cytokeratin djacent Cancer associated fibroblasts () C a a a α-sm ctin Patient # ig. S9: Characterization of the patient-derived fibroblasts., Phase contrast images of primary human normal omental fibroblasts (s), adjacent s (as) and cancer-associated fibroblasts (s)., Immunofluorescence staining (green) of vimentin, α-smooth muscle actin (α-sm) and cytokeratinin s, adjacent s and s. ll cells were counterstained with DPI (blue). C, Immunoblotting for α-smooth muscle actin (α-sm) in s and adjacent s (as) in three different patients. β-actin (actin) was used as a loading control. Page of