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Supplemental Figure Legend
Supplemental Figure 1
(A) Principal component analysis of gene expression of Panc-1 and BxPC3 cells. All
controls: [Ctrl: untreated cells, Ctrl Exo: control exosomes (no siRNA payload), Scr
(siScrbl Exo), sirnaExo (exosomes with KrasG12D siRNA payload)]. (B-E) Heat map
depicting expression of top 1,000 genes (B-D) and differentially expressed genes
(statistically significant) (C-E) in Panc-1 (B, C) and BxPC3 (D, E) cells. Red: sirnaExo:
exosomes with KrasG12D siRNA payload, black: Scr: siScrl Exo, dark grey: CtrlExo: control
exosomes (no siRNA payload), light grey: Ctrl (untreated cells). (F) Gene set enrichment
plot for significantly down and up-regulated genes in siKrasG12D-1 Exo treated Panc-1 cells
compared to all controls.
Supplemental Figure 2
(A) Sanger sequencing of PDX tumors (n=2 mice), to confirm the G to A base substitution
associated with the glycine to aspartic acid mutation at codon 12 (G12D). (B) Comparison
of quantification of tumor burden pre-treatment (baseline) by ultrasound (US, day 58 post
tumor induction), and MRI (day 60 post tumor induction), siKrasG12D-1 iExo (n=7) and
siScrbl iExo (n=7). Each set of bars for US and MRI represents measures for individual
mice. (C) Detailed findings from individual PDX mouse treated with siKrasG12D-1 iExo (n=7)
and siScrbl iExo (n=7), showing survival post treatment start (PTS) and post birth (PB),
baseline pre-treatment tumor volumes as measure by both ultrasound (US) and MRI,
post-treatment MRI measurements of tumor volume, tumor weight (TW) and body weight
(BW) at end point, and observations at necropsy/euthanasia. No measurements for MRI
was recorded if the mice were euthanized or the imaging was of poor quality (CNBM:
could not be measured). N/A: not applicable. (D) Pictures of H&E stained tissue sections
showing lesions of inflammation observed in organs of a siKrasG12D-1 iExo treated mouse
(A1-B). See accompanying source data.
Supplemental Figure 3
(A) Number of exosomes per cell per hour determine by NanoSightTM for each MSC
donors listed. Donor 3 was used for all subsequent MSCs-derived exosomes studies.
Three independent measurements from distinct conditioned media collection are shown.
(B-C) Flow cytometry analyses histogram plots of the listed cell surface markers for BJ
fibroblast cells (B) and mesenchymal stem cells, MSCs (C). The inset numbers depict the
percent positive cells. In grey: isotype control. See accompanying source data.
Supplemental Figure 4
(A) Schematic detailing the procedure for MSCs bioreactor culture for collection of
exosomes (B) Schematic of the isolation and electroporation procedures of exosomes
from MSCs conditioned media. (C) Schematic depicting the generation of MSCs-derived
iExosomes.
Supplemental Figure 5
(A) Tabulated data depicting the quantitation of total exosomal protein and number of
exosomes in each of the 6 consecutive 48 hours harvests of 250 ml MSCs conditioned
media from the bioreactor. The data are also depicted in Figure 3A-C. (B) Quantitation of
the number of exosomes produced from three distinct bioreactors. (C) Quantitation of the
levels of glucose and lactose in the bioreactor, for the total duration of bioreactor
experiment (21 days). (D) Representative histogram of flow cytometry analysis of
exosomal markers (CD9, CD63, CD81) and CD47 on exosomes from all bioreactor
harvests (Bioreactor harvests 1 and 6 data are also shown in Figure 3E). Inset numbers
represent the percent positive beads. In grey: isotype control. See accompanying source
data.
Supplemental Figure 6
(A) Quantification of apoptosis assayed by flow cytometry analyses in 689KPC cells (n=2
distinct experiments). One-way ANOVA comparing experimental groups to untreated. (B)
Images of H&E stained tissue sections showing representative lesions observed in the
pancreas, liver, spleen and kidney of KPC689 mice at experimental endpoint. Control Exo
(n=4), BJ siKrasG12D-1 iExo (n=6) and MSCs siKrasG12D-1 iExo (n=6). Scale bar: 100 μm.
(C) Percent mice with macroscopic metastases in the indicated organs at time of
necropsy. Control Exo (n=4), BJ siKrasG12D-1 iExo (n=6) and MSCs siKrasG12D-1 iExo
(n=6). (D) Detailed findings from individual mice with KPC689 tumors treated with Control
Exo (n=4), BJ siKrasG12D-1 iExo (n=6) and BJ siKrasG12D-1 iExo (n=6), showing survival
post treatment start (PTS) and post birth (PB), tumor weight (TW) and body weight (BW),
number of lung macrometastases, and observations at necropsy/euthanasia. (E) Tumor
burden (%) of KPC689 mice at experimental endpoint. Control Exo (n=4), BJ siKrasG12D-
1 iExo (n=6) and MSCs siKrasG12D-1 iExo (n=6). (F) Tumor weights (g: grams) of KPC689
mice at experimental endpoint. Control Exo (n=4), BJ siKrasG12D-1 iExo (n=6) and MSCs
siKrasG12D-1 iExo (n=6). (G) KPC689 orthotopic tumor growth (bioluminescence), and
average total flux at day 51, post tumor induction. Control Exo (n=4), BJ siKrasG12D-1 iExo
(n=6) and MSCs siKrasG12D-1 iExo (n=6). The mean +/- SEM is depicted and statistical
tests are listed for relevant panels. ** p<0.01. See accompanying source data.
Supplemental Figure 7
(A) Detailed findings from individual mice with Panc-1 tumors treated with Control Exo
(n=4), BJ siKrasG12D-1 iExo (n=6) and BJ siKrasG12D-1 iExo (n=6), showing survival post
treatment start (PTS) and post birth (PB), tumor weight (TW) and body weight (BW), and
observations at necropsy/euthanasia. ** this tumor weight was not included; the pancreas
of this mouse was found grossly and histopathologically normal. (B) Pictures of H&E
stained sections of tumors from Panc-1 tumor bearing mice. Control Exo (n=5), MSCs
siKrasG12D-1 iExo, CB (n=5), MSCs siKrasG12D-1 iExo, RB (n=4), BJ siKrasG12D-1 iExo, RB
(n=5). Scale bars: 100 μm (top) and 50 μm (bottom). These data are also depicted in
Figure 4O. (C) Representative pictures of luciferase activity of Panc-1 orthotopic tumors
at day 35 and day 84, post tumor induction. Control Exo (n=5), MSCs siKrasG12D-1 iExo,
CB (n=5), MSCs siKrasG12D-1 iExo, RB (n=4), BJ siKrasG12D-1 iExo, RB (n=5). (D) Panc-1
orthotopic tumor growth (bioluminescence) over time. Control Exo (n=5), MSCs
siKrasG12D-1 iExo, CB (n=5), MSCs siKrasG12D-1 iExo, RB (n=4), BJ siKrasG12D-1 iExo, RB
(n=5). (E) Tumor burden (%) of Panc-1 tumor bearing mice at experimental endpoint.
Control Exo (n=5), MSCs siKrasG12D-1 iExo, CB (n=5), MSCs siKrasG12D-1 iExo, RB (n=4),
BJ siKrasG12D-1 iExo, RB (n=5). (F) Tumor weights (g: grams) of Panc-1 tumor bearing
mice at experimental endpoint. Control Exo (n=5), MSCs siKrasG12D-1 iExo, CB (n=5),
MSCs siKrasG12D-1 iExo, RB (n=4), BJ siKrasG12D-1 iExo, RB (n=5). (G) Flow cytometry
analyses and quantification of apoptosis in Panc-1 cells comparing the efficacy of 48
distinct electroporation conditions (A1-A16; B1-B16; C1-C16) of MSCs and BJ Fibroblast
siKrasG12D iExosomes, using a large volume electroporation system and CB (n=2 distinct
experiments). The electroporation condition subsequently chosen was A-14. The mean
+/- SEM is depicted. See accompanying source data.
Supplemental Figure 8
(A) Schematic representation of the experiment using KPC689 orthotopic tumor bearing
mice (related to experiment in Figure 5E). (B) Comparison of baseline tumor
bioluminescence (total Flux) pre-treatment, of KPC689 orthotopic experiments 1 and 2,
to show baseline tumor measurements pre-treatment of Experiment-2 was higher than
that of Experiment-1. Experiment-1: Control Exo (n=4), BJ siKrasG12D-1 iExo (n=6) and
MSCs siKrasG12D-1 iExo (n=6). Experiment-2: CB/PBS (n=7), Control Exo (n=7),
Gemcitabine (n=8), MSCs siKrasG12D-2 iExo (n=8), Gemcitabine + MSCs siKrasG12D-2 iExo
(n=8). Two-tailed unpaired t test between experiment 1 and 2. (C) Detailed findings from
individual mice with KPC689 tumors (Experiment-2) treated with CB/PBS (n=7), Control
Exo (n=7), Gemcitabine (n=8), MSCs siKrasG12D-2 iExo (n=8), Gemcitabine + MSCs
siKrasG12D-2 iExo (n=8), showing survival post treatment start (PTS) and post birth (PB),
tumor weight (TW) and body weight (BW), number of lung macrometastases, and
observations at necropsy/euthanasia. (D) Representative images of H&E stained
sections of pancreatic tumors from KPC689 tumor bearing mice. Scale bars 100 μm (top)
and 50 μm (bottom). CB/PBS (n=7), Control Exo (n=7), Gemcitabine (n=8), MSCs
siKrasG12D-2 iExo (n=8), Gemcitabine + MSCs siKrasG12D-2 iExo (n=8). The mean +/- SEM
is depicted. See accompanying source data.
Supplemental Figure 9
(A) Schematic representation of the experiment using KPC689 orthotopic tumor bearing
mice (related to experiment in Figure 5F). (B) Detailed findings from individual mice with
KPC689 tumors treated with CB/PBS (n=7), Control Exo (n=7), Gemcitabine (n=7), MSCs
siKrasG12D-2 iExo (n=7), Gemcitabine + MSCs siKrasG12D-2 iExo (n=7), showing survival
post treatment start (PTS) and post birth (PB), tumor weight (TW) and body weight (BW),
number of lung macrometastases, and observations at necropsy/euthanasia.
Supplemental Figure 10
(A) Cytotoxicity tests performed on the blood collected from adult C57Bl/6J mice injected
i.p. every other day for 120 days. PBS (n=3), BJ fibroblasts exosomes (n=4). ALT: alanine
aminotransferase; AST: aspartate aminotransferase; BUN: blood urea nitrogen. The grey
highlight defines the normal range. (B) Representative pictures of H&E stained tissue
sections showing lesions of inflammation observed in the mesenteric lymph node, kidney,
liver and gall-bladder.
Supplemental Figure 11
(A) Immunotyping analyses of spleen, bone marrow and thymus of immunocomptent non-
tumor bearing mice administered with the indicated treatments. ‡: these values are not
erroneous despite their similarities. #: this group shows statistical significant differences
with exosomes treated mice but not with CB. (B) ELISA for IL-6 in the indicated groups.
0 denotes that the levels were below the limits of the assay (not detected).
Supplemental Figure 12
(A) Biodistribution in the indicated organs of DiR labeled MSCs exosomes 6 hours post
i.v. injection in nude mice (left) or 3 hours post i.v. injection in tumor bearing (KPC689)
C57BL/6 mice. DiR only (n=1), DiR labeled MSCs exosomes (n=2). (B) Biodistribution in
the indicated organs of DiR labeled MSCs exosomes 3 hours post i.p. or i.v. injection of
C57BL/6 non-tumor bearing mice. (C) Biodistribution in the indicated organs of DiR
labeled MSCs exosomes 24 and 48 hours post i.p. injection tumor bearing (KPC689)
C57BL/6 mice. These tumors are depicted in Figure 7B.
Supplemental Figure 13
(A) Representative dot plot of flow cytometry analyses of apoptosis in Panc-1 cells
induced by MSCs siKrasG12D-2 iExosomes freeze/thaw during 3 and 6 months showing
similar efficiency that fresh iExosomes. n=2 independent experiments, quantitation is
shown in Figure 6I. (B) Detailed findings from individual PKS mice treated with MSCs
siKrasG12D-2 iExo (n=4) and Control Exo (n=5), showing treatment groups, start of
treatment, survival of mice, body weight (BW) and tumor weight (TW) at experimental
endpoint. See accompanying source data.
Supplementary Table 1
Detailed necropsy analyses of C57BL/6 mice injected i.p. with either empty BJ Fibroblast
exosomes (n=4) or PBS (n=3), every other day, for 120 days.
Supplemental Table 2
Summary of treatments for the in vivo experiments.
Supplementary Table 3
Detailed information on the antibodies used in this study.
Supplemental Figure 1
Panc−
1 iExo−3
Panc−
1 iExo−2
Panc−
1 iExo−1
Panc−
1 con
t−3
Panc−
1 scr−
2
Panc−
1 scr−
1
Panc−
1 con
t−1
Panc−
1 CE−
1
Panc−
1 CE−
3ILMN_1701789ILMN_3242144ILMN_1693483ILMN_2239754ILMN_1791280ILMN_3241369ILMN_3240150ILMN_2165762ILMN_2295879ILMN_1705750ILMN_3240306ILMN_1770994ILMN_1661470ILMN_1716264ILMN_1774973ILMN_1743217ILMN_3244592ILMN_3238478ILMN_1766650ILMN_1662358ILMN_3241140ILMN_1734542ILMN_1658247ILMN_2053527ILMN_1739428ILMN_1795963ILMN_1665554ILMN_1874689ILMN_1724533ILMN_1814305ILMN_1891455ILMN_1682354ILMN_1782167ILMN_2376403ILMN_3248562ILMN_1770030ILMN_1652434ILMN_1754894ILMN_1885233ILMN_1784380ILMN_3247008ILMN_1689294ILMN_1713832ILMN_1730201ILMN_2214355ILMN_3235397ILMN_3241139ILMN_1652638ILMN_1747183ILMN_1660900ILMN_1711120ILMN_1731224ILMN_1698191ILMN_1811866ILMN_1682636ILMN_1741632ILMN_1704056ILMN_1789990ILMN_2370624ILMN_3274045ILMN_1699772ILMN_3241798ILMN_1666757ILMN_1762275ILMN_1728106ILMN_3243644ILMN_1760102ILMN_1795317ILMN_1795338ILMN_1740555ILMN_1794560ILMN_2112730ILMN_1762606ILMN_1667594ILMN_1654429ILMN_1711838ILMN_1739103ILMN_1701487ILMN_3233229ILMN_2181892ILMN_1665832ILMN_1659953ILMN_1810127ILMN_1693401ILMN_2401701ILMN_1711005ILMN_1694367ILMN_1669700ILMN_2209515ILMN_2377829ILMN_1766054ILMN_1911605ILMN_1657348ILMN_2052373ILMN_1745807ILMN_1696846ILMN_1672295ILMN_1675992ILMN_1693242ILMN_1785037ILMN_1787539ILMN_3249167ILMN_1745397ILMN_1810712ILMN_1713351ILMN_1709604ILMN_2376205ILMN_1670970ILMN_1802096ILMN_3220934ILMN_1797596ILMN_1654072ILMN_1701918ILMN_1813685ILMN_1762003ILMN_1867149ILMN_1745148ILMN_1659544ILMN_2123431ILMN_1781373ILMN_1780711ILMN_1685703ILMN_2401618ILMN_1764383ILMN_1806845ILMN_1716265ILMN_1764052ILMN_3237291ILMN_1690125ILMN_1778625ILMN_1741727ILMN_1659463ILMN_1678919ILMN_1683415ILMN_1788832ILMN_3240586ILMN_3251232ILMN_2393046ILMN_1744006ILMN_1655748ILMN_1728083ILMN_1859908ILMN_1690464ILMN_3307892ILMN_1699525ILMN_2381697ILMN_2089167ILMN_3237977ILMN_1771697ILMN_1809483ILMN_1696956ILMN_1772292ILMN_1863592ILMN_1681644ILMN_1654685ILMN_1879482ILMN_1695606ILMN_1667239ILMN_1764851ILMN_2214678ILMN_1764723ILMN_1854833ILMN_1758337ILMN_1722292ILMN_1722781ILMN_1783185ILMN_2388800ILMN_1765274ILMN_2160160ILMN_1738725ILMN_3204890ILMN_1756417ILMN_3234762ILMN_1710544ILMN_1695246ILMN_3200140ILMN_2198878ILMN_1777483ILMN_1712298ILMN_3244157ILMN_1766219ILMN_1703913ILMN_1764166ILMN_1754332ILMN_1815705ILMN_1696622ILMN_1748124ILMN_1657234ILMN_1745154ILMN_2400219ILMN_1803611ILMN_2415144ILMN_2067852ILMN_1716441ILMN_3235185ILMN_2413158ILMN_1795778ILMN_2318685ILMN_2283325ILMN_1680465ILMN_1713846ILMN_1709287ILMN_1652762ILMN_3249435ILMN_2082762ILMN_1714765ILMN_1763000ILMN_1800733ILMN_3242448ILMN_1671837ILMN_1837935ILMN_1708110ILMN_1727855ILMN_2170625ILMN_1756784ILMN_3310840ILMN_1701111ILMN_2226628ILMN_2303955ILMN_1760556ILMN_1798181ILMN_3248781ILMN_1688103ILMN_1856480ILMN_1707051ILMN_1659976ILMN_1712545ILMN_1814151ILMN_1758164ILMN_1792110ILMN_3238283ILMN_3251634ILMN_2348268ILMN_1661363ILMN_1723706ILMN_1693259ILMN_1677376ILMN_1855278ILMN_1754860ILMN_1656129ILMN_1743402ILMN_3265237ILMN_1690442ILMN_1674399ILMN_1663119ILMN_1752478ILMN_1713751ILMN_2149164ILMN_3188106ILMN_1802404ILMN_1705241ILMN_1774265ILMN_1804929ILMN_1706118ILMN_1745420ILMN_1705032ILMN_1745374ILMN_1715814ILMN_2229877ILMN_1733615ILMN_1800942ILMN_1800512ILMN_1669210ILMN_1708778ILMN_1707695ILMN_2063584ILMN_1763745ILMN_2386205ILMN_2385278ILMN_1681984ILMN_1779376ILMN_3182171ILMN_1746673ILMN_2220184ILMN_1745116ILMN_2130514ILMN_2322375ILMN_1662243ILMN_1765825ILMN_1677691ILMN_1779356ILMN_1746819ILMN_1682658ILMN_1666306ILMN_1667825ILMN_2054297ILMN_1747673ILMN_1807873ILMN_1691790ILMN_1710000ILMN_2124155ILMN_3251691ILMN_1718309ILMN_2339705ILMN_2218758ILMN_1704529ILMN_2327795ILMN_1815707ILMN_1678808ILMN_1660079ILMN_1680692ILMN_1784749ILMN_1702691ILMN_2092756ILMN_1810797ILMN_1673548ILMN_1745079ILMN_1729748ILMN_2381296ILMN_1903159ILMN_1701724ILMN_3294134ILMN_1703891ILMN_3251550ILMN_1696946ILMN_3247261ILMN_1706873ILMN_1695590ILMN_2086077ILMN_2148012ILMN_1700231ILMN_2173835ILMN_2347798ILMN_1717639ILMN_2377980ILMN_1667748ILMN_1804051ILMN_1678669ILMN_1765966ILMN_2220403ILMN_1684197ILMN_1814966ILMN_2353642ILMN_1774432ILMN_1763144ILMN_1721833ILMN_1807136ILMN_1785284ILMN_1738712ILMN_1680347ILMN_2179726ILMN_1680364ILMN_1679133ILMN_1787509ILMN_1772959ILMN_2135175ILMN_1746801ILMN_1719975ILMN_1792409ILMN_1779043ILMN_1792910ILMN_3308753ILMN_1724789ILMN_3187328ILMN_1667711ILMN_1667201ILMN_3282587ILMN_2389376ILMN_3298694ILMN_1680091ILMN_1710284ILMN_2348975ILMN_2213199ILMN_1680139ILMN_1697153ILMN_1793549ILMN_1701386ILMN_1713901ILMN_1747556ILMN_1677466ILMN_1729749ILMN_1656574ILMN_2268068ILMN_2327346ILMN_1745034ILMN_1660871ILMN_1779751ILMN_1682812ILMN_1710075ILMN_2183409ILMN_1659937ILMN_1806486ILMN_1779558ILMN_2204754ILMN_1660718ILMN_1680693ILMN_1726104ILMN_1809842ILMN_1659027ILMN_1760121ILMN_1728071ILMN_1717313ILMN_1812926ILMN_3247018ILMN_2134538ILMN_3306173ILMN_2392286ILMN_2198515ILMN_1687867ILMN_1730809ILMN_2325347ILMN_1704385ILMN_1815733ILMN_1788213ILMN_1736847ILMN_1812441ILMN_1773742ILMN_1810069ILMN_1724406ILMN_2390310ILMN_2374115ILMN_1781155ILMN_3190972ILMN_1795826ILMN_1786347ILMN_2246510ILMN_1662065ILMN_3229552ILMN_3310416ILMN_1783156ILMN_1734814ILMN_1674038ILMN_1892638ILMN_1804988ILMN_1756676ILMN_1756999ILMN_3241091ILMN_1673275ILMN_1719286ILMN_1692191ILMN_2120022ILMN_1737949ILMN_1813400ILMN_1665291ILMN_1740604ILMN_1778173ILMN_1790537ILMN_2209578ILMN_1745282ILMN_1774800ILMN_1789384ILMN_1703430ILMN_1667839ILMN_1669584ILMN_1677092ILMN_1696254ILMN_2374159ILMN_1798212ILMN_1702065ILMN_1715324ILMN_1666776ILMN_2367883ILMN_1731720ILMN_2383383ILMN_1660111ILMN_1776047ILMN_2355033ILMN_3233179ILMN_1721704ILMN_1788547ILMN_2119555ILMN_3268697ILMN_1694686ILMN_2075927ILMN_1674302ILMN_2328666ILMN_1803317ILMN_3227023ILMN_1726786ILMN_3244640ILMN_2166524ILMN_1736042ILMN_2231021ILMN_3211887ILMN_1773865ILMN_1673936ILMN_1695475ILMN_1707748ILMN_1683096ILMN_1786041ILMN_2374778ILMN_1700202ILMN_1763228ILMN_1749011ILMN_1723768ILMN_1812297ILMN_2381064ILMN_1794539ILMN_1701131ILMN_3235326ILMN_1693341ILMN_1802205ILMN_1676191ILMN_1685781ILMN_1781636ILMN_1671005ILMN_1799744ILMN_1737604ILMN_1786976ILMN_1720270ILMN_1802615ILMN_2285802ILMN_1806907ILMN_1732071ILMN_2358540ILMN_1664216ILMN_1700584ILMN_1807596ILMN_1663042ILMN_1701457ILMN_1743836ILMN_1794875ILMN_1728228ILMN_1775822ILMN_1735930ILMN_1665865ILMN_1785336ILMN_1791569ILMN_2143155ILMN_1696643ILMN_1746883ILMN_1748827ILMN_1802380ILMN_1705814ILMN_1719870ILMN_1718898ILMN_1734833ILMN_1678904ILMN_1669252ILMN_1717934ILMN_1656196ILMN_1696432ILMN_1705093ILMN_1790534ILMN_1669905ILMN_2220187ILMN_1802355ILMN_1756806ILMN_1661595ILMN_2360730ILMN_1798886ILMN_2374164ILMN_1663866ILMN_1726288ILMN_1664303ILMN_1720858ILMN_1771333ILMN_3308961ILMN_1771841ILMN_1664068ILMN_2148847ILMN_1655469ILMN_1719695ILMN_1792681ILMN_1745471ILMN_1667213ILMN_1794501ILMN_1794726ILMN_1732688ILMN_1780582ILMN_1681737ILMN_1713764ILMN_2154322ILMN_2385220ILMN_1717056ILMN_1693620ILMN_1700690ILMN_2405018ILMN_1690105ILMN_1780302ILMN_1659122ILMN_1770811ILMN_1689378ILMN_1763837ILMN_1753183ILMN_1756992ILMN_2157510ILMN_1898723ILMN_1682799ILMN_1684289ILMN_1664466ILMN_2147114ILMN_1787815ILMN_1737728ILMN_2059294ILMN_2386100ILMN_1761247ILMN_1729033ILMN_1677814ILMN_1726460ILMN_2376408ILMN_1691111ILMN_2214997ILMN_1660554ILMN_1657153ILMN_1671603ILMN_1728972ILMN_1792455ILMN_1807719ILMN_1787844ILMN_1668012ILMN_1698209ILMN_1790549ILMN_1683891ILMN_1666733ILMN_2411897ILMN_1664912ILMN_1685916ILMN_1748018ILMN_1682775ILMN_1655990ILMN_1671557ILMN_1777794ILMN_1808354ILMN_3223500ILMN_1661599ILMN_1792138ILMN_1793616ILMN_1679093ILMN_1683859ILMN_1670752ILMN_1787897ILMN_1699354ILMN_3237404ILMN_1787691ILMN_1764500ILMN_2086105ILMN_3231881ILMN_3214893ILMN_2146372ILMN_2150465ILMN_1687375ILMN_2122511ILMN_2186061ILMN_2155228ILMN_1687583ILMN_1793672ILMN_1758658ILMN_1712112ILMN_3270641ILMN_1698148ILMN_1838313ILMN_1729234ILMN_1724145ILMN_1675448ILMN_1700276ILMN_1675239ILMN_3227315ILMN_1745806ILMN_1711702ILMN_2320250ILMN_3237177ILMN_1715401ILMN_2415979ILMN_1667430ILMN_1720158ILMN_1815319ILMN_1679405ILMN_1735453ILMN_1732452ILMN_1757497ILMN_1651705ILMN_1661593ILMN_1804610ILMN_1669046ILMN_1665455ILMN_1668507ILMN_1717674ILMN_2045994ILMN_1701998ILMN_2084353ILMN_1805519ILMN_2173294ILMN_2338963ILMN_2397954ILMN_2350574ILMN_3251737ILMN_1791576ILMN_3238680ILMN_3274596ILMN_2404135ILMN_3239861ILMN_2222008ILMN_1803988ILMN_1755909ILMN_1793476ILMN_3248773ILMN_1808938ILMN_1727194ILMN_1674551ILMN_3197767ILMN_1815308ILMN_1758811ILMN_2054019ILMN_1759954ILMN_1768973ILMN_3181480ILMN_1763688ILMN_2398107ILMN_3288717ILMN_2181432ILMN_1751072ILMN_2219618ILMN_1777740ILMN_3279414ILMN_1719039ILMN_1703697ILMN_1746525ILMN_1652379ILMN_2144426ILMN_3272603ILMN_2091347ILMN_3305304ILMN_1726783ILMN_1737314ILMN_1682738ILMN_1768101ILMN_1814122ILMN_2231020ILMN_1898124ILMN_1664516ILMN_1681016ILMN_1672504ILMN_1706502ILMN_3229324ILMN_2110908ILMN_1671554ILMN_2180371ILMN_1776109ILMN_1796074ILMN_1778836ILMN_1666453ILMN_1796177ILMN_1654920ILMN_2380588ILMN_1775304ILMN_1780236ILMN_1708006ILMN_2096784ILMN_1770053ILMN_2224907ILMN_1668721ILMN_3206804ILMN_2365465ILMN_3305938ILMN_1658847ILMN_1677607ILMN_1716446ILMN_1662340ILMN_2353358ILMN_1695706ILMN_1756220ILMN_2364521ILMN_2057573ILMN_2367239ILMN_1664756ILMN_1777325ILMN_2294762ILMN_1740938ILMN_1679796ILMN_1794017ILMN_1723971ILMN_1693270ILMN_2374692ILMN_1652128ILMN_1752631ILMN_2342437ILMN_1703946ILMN_2192694ILMN_1790778ILMN_1669523ILMN_2061565ILMN_2348788ILMN_1811104ILMN_2136089ILMN_1672004ILMN_1791896ILMN_1746408ILMN_2089329ILMN_1707503ILMN_1653856ILMN_1755405ILMN_1674661ILMN_1755354ILMN_1814971ILMN_1797964ILMN_1669113ILMN_1708375ILMN_1791296ILMN_2215382ILMN_1744381ILMN_1805192ILMN_1698213ILMN_1669456ILMN_1699856ILMN_1812769ILMN_1706013ILMN_1733519ILMN_3242900ILMN_1695827ILMN_2313889ILMN_2339955ILMN_1803824ILMN_3289352ILMN_1720282ILMN_1772821ILMN_3310351ILMN_1654268ILMN_1750278ILMN_1778611ILMN_1660793ILMN_1673113ILMN_1738150ILMN_1703123ILMN_3239181ILMN_1696911ILMN_2061435ILMN_1700681ILMN_2356654ILMN_1747016ILMN_1718063ILMN_1732296ILMN_1659047ILMN_2285713ILMN_1784709ILMN_1759915ILMN_1776105ILMN_2092536ILMN_2405684ILMN_1673305ILMN_3268165ILMN_1809433ILMN_2113738ILMN_1763091ILMN_1719749ILMN_1780058ILMN_1737588ILMN_1706015ILMN_1691364ILMN_2230902ILMN_1740426ILMN_1727479ILMN_2125675ILMN_2349459ILMN_1687384ILMN_1664861ILMN_2099301ILMN_1674620ILMN_1658289ILMN_1801939ILMN_1702487ILMN_2070052ILMN_2049727ILMN_1791226ILMN_2094776ILMN_3239135ILMN_2200917ILMN_1760667ILMN_2286014ILMN_1702168ILMN_1692145ILMN_1778561ILMN_1797776ILMN_1776080ILMN_1671893ILMN_2063168ILMN_2136133ILMN_1782305ILMN_1659936ILMN_1805737ILMN_2395043ILMN_2041190ILMN_1803811ILMN_1695420ILMN_1685480ILMN_2073235ILMN_1720889ILMN_3214256ILMN_2404850ILMN_2285996ILMN_3205271ILMN_1701877ILMN_3304111ILMN_1776181ILMN_1665823ILMN_2396020ILMN_1737406ILMN_2184373ILMN_2347068ILMN_1776347ILMN_1763540ILMN_1658835ILMN_1749662ILMN_1680618ILMN_1707727ILMN_3305169ILMN_1806692ILMN_2194852ILMN_1655710ILMN_2051900ILMN_1656501ILMN_3206343ILMN_1775170ILMN_3187612ILMN_3244117ILMN_2188119ILMN_1795930ILMN_1683664ILMN_1684306ILMN_1786242ILMN_1761804ILMN_3202024ILMN_1784602ILMN_1795937ILMN_1687887ILMN_3251472ILMN_2134381ILMN_2408415ILMN_2345837ILMN_1742163ILMN_3237656ILMN_1776052ILMN_2091123ILMN_1803398ILMN_1688780ILMN_1804396ILMN_1748591ILMN_1706687ILMN_2041046ILMN_2192693ILMN_3225591ILMN_1796417ILMN_1736597ILMN_3278506ILMN_1811468ILMN_1737184ILMN_1757467ILMN_1673711ILMN_1769705ILMN_3178792ILMN_1713892ILMN_1656057ILMN_3264112ILMN_1668052ILMN_1688480ILMN_1765446ILMN_1668411ILMN_1773337ILMN_1806023ILMN_1711862ILMN_2313730ILMN_2115125ILMN_3275345ILMN_2094106ILMN_1724040ILMN_1731619ILMN_2130078ILMN_2167922ILMN_1711823ILMN_1743199ILMN_1756326ILMN_1805028ILMN_3208715ILMN_1781285ILMN_1762899ILMN_1660577ILMN_2058141ILMN_1716678ILMN_1735014ILMN_1746252ILMN_1689327ILMN_1869109ILMN_3299478ILMN_1697469ILMN_2156267ILMN_1682717ILMN_1751607ILMN_2149226ILMN_1687978ILMN_1675848ILMN_2234016ILMN_1665510ILMN_2308849ILMN_3300797ILMN_1713636ILMN_3302177ILMN_1768534ILMN_2060413ILMN_1773154ILMN_1803429ILMN_1814823ILMN_3176403ILMN_2188264ILMN_1699829ILMN_1740429ILMN_1686664ILMN_1691156ILMN_1753342ILMN_1720829ILMN_1770338
−2 0 1 2Row Z−Score
Color Key
BXPC−3 s
cr−1
BXPC−3 s
cr−3
BXPC−3 s
cr−2
BXPC−3 c
ont−3
BXPC−3 c
ont−1
BXPC−3 c
ont−2
BXPC−3 i
EXO−3
BXPC−3 C
E−1
BXPC−3 C
E−2
BXPC−3 C
E−3
BXPC−3 i
EXO−2
BXPC−3 i
EXO−1
−2 0 2Row Z−Score
Color Key
BXPC
−3 C
E−1
BXPC
−3 s
cr−3
BXPC
−3 s
cr−2
BXPC
−3 s
cr−1
BXPC
−3 c
ont−
3
BXPC
−3 c
ont−
2
BXPC
−3 c
ont−
1
BXPC
−3 C
E−2
BXPC
−3 C
E−3
BXPC
−3 iE
XO−2
BXPC
−3 iE
XO−1
BXPC
−3 iE
XO−3
−2 0 1 2Row Z−Score
Color Key
Panc−1
iExo−3
Panc−1
iExo−2
Panc−1
iExo−1
Panc−1
con
t−3
Panc−1
scr−2
Panc−1
scr−1
Panc−1
con
t−1
Panc−1
CE−
1
Panc−1
CE−
3
−2 0 1 2Row Z−Score
Color Key
A D
C E
F
NES=2.038FDR=0.0053
NES=2.0382FDR=0.0058
NES=2.034FDR=0.0065
NES=-2.0005FDR=0.0096 NES=-2.0629
FDR=0.0026
NES=-2.2036FDR≃0.0
NES=-2.234FDR≃0.0
NES=-2.0402FDR=0.0039
BxPC-3
−0.2 −0.1 0.0 0.1 0.2 0.3
−0.1
5−0
.10
−0.0
50.
000.
050.
10
Leading logFC dim 1
Lead
ing
logF
C d
im 2
BXPC3_Scr
BXPC3_Scr
BXPC3_Scr
BXPC3_CtrlExoBXPC3_CtrlExo
BXPC3_CtrlExo
BXPC3_sirnaExo
BXPC3_sirnaExo
BXPC3_sirnaExo
BXPC3_Ctrl
BXPC3_CtrlBXPC3_Ctrl
Panc-1
−0.4 −0.2 0.0 0.2 0.4 0.6 0.8
−0.1
0.0
0.1
0.2
0.3
Leading logFC dim 1
Lead
ing
logF
C d
im 2
Panc1_Scr
Panc1_Scr
Panc1_CtrlExo
Panc1_CtrlExoPanc1_sirnaExo
Panc1_sirnaExo
Panc1_sirnaExo
Panc1_Ctrl
Panc1_Ctrl
NES=2.1315FDR=0.0019
NES=2.1518FDR=0.0022
NES=2.1214FDR=0.0017
B
Supplemental Figure 2
Bowel Kidney Lung
Spleen Liver Pancreas
G G T
G A/G T
G A/G T
Tumor A2-rsi
Tumor B1-0scr0
200
400
600
Tum
or v
olum
e (m
m3 )
siScrbl siKrasG12D-1
US
MRI
A BM
ouse
1M
ouse
2
C
D
Survival (Days) Pre-treatment Post-treatment Post-treatment Post-treatment End-point Observations Notes
Mouse ID Treatment PTS PB Tumor volume (mm3) (US)
Tumor volume (mm3) (MRI)
BW(g) MRI-2 MRI-3 MRI-4 TW(g) BW(g) Necropsy notesSuspected
tumor burden related death
A1-Y siKrasG12D iExo 133 286 211.87 239.56 23.4 CNBM CNBM Euthanized 0.87 28.8 Jaundice, very small tumor burden No
A1-B siKrasG12D iExo 316 469 94.49 112.27 24.6 415.44 146.24 26.98 0.38 31.9 Hunched, swollen intestine, kidney atrophy, mesenteric abscess, minimal tumor burden
No
A1-G siKrasG12D iExo 257 410 86.84 108.52 22.1 515.05 126.97 10.38 0.2 23.8 Hunched, kidney atrophy, minimal tumor buden No
A1-O siKrasG12D iExo 360 513 100.08 167.31 25.3 363.45 150.16 63.84 0.24 24.2Enlarged mesenteric lymph node, minimal tumor
burden No
A1-W siKrasG12D iExo 254 407 225.92 231.50 23.6 CNBM 212.13 50.44 0.28 28.1Enlarged eyes, euthanasia due to neurological
problems, small tumor No
A2-Y siKrasG12D iExo 35 188 434.96 481.62 22.1 642.22 Euthanized Euthanized 1.4 29.2 Moderate tumor burden, euthanized as an age matched comparison to B1-O
N/A: age-matched
A2-NT siKrasG12D iExo 178 331 212.71 206.30 25.1 CNBM 965.71 CNBM 1.8 32.2Small primary tumor, large secondary tumor,
euthanized due to tumor burden Yes
Average: 195.27 221.01 484.04 320.24 37.91 0.74
B1-Y siScrbl iExo 206 359 158.60 186.11 25.0 CNBM 654.78 Euthanized 1.21 26.1Moderate tumor burden, hunched, scaly skin at
euthanasia Yes
B1-B siScrbl iExo 168 321 114.13 112.30 24.4 773.19 805.08 Euthanized 1.61 26.0 Moderate tumor burden, weak, swollen bowel YesB1-G siScrbl iExo 144 297 98.81 146.54 23.1 961.41 1977.47 Euthanized 5.84 27.9 Extensive tumor burden, hunched posture, weak Yes
B1-O siScrbl iExo 35 188 447.43 379.32 22.9 1254.40 Euthanized Euthanized 2.3 31.2Extensive tumor burden, euthanized due to tumor
burden, aged matched with A2-Y Yes
B1-W siScrbl iExo 102 255 176.84 182.64 24.8 CNBM 2225.12 Euthanized 4.67 34.1Large primary and secondary tumors, spleen
invasion, macroscopic liver nodulesN/A: age-matched
B2-Y siScrbl iExo 158 311 250.26 263.35 26.1 950.14 CNBM Euthanized 2.4 28.8 Extensive tumor burden, weak, hunched posture YesB2-NT siScrbl iExo 69 222 86.75 120.45 25.4 CNBM 969.73 Euthanized 1.69 29.2 Weak, moderate tumor burden, spleen atrophy Yes
Average: 190.40 198.68 984.79 1326.43 2.82
Supplemental Figure 3
100% 100% 96%
100%
100%
0.8% 0.1% 0.2%
47%86%
100%100%
0.2%
84%
3.6%
100% 100% 81%
0.1%
94%
83%
100% 100% 93%
99%
98.1%
99%
3.6% 0.5%0%
B C
CD44 CD90 CD105
CD73 HLA-ABCCD29
CD9 CD63CD81
CD47 CD45CD10
HLA-DR CD34CD31 HLA-DR CD34CD31
CD47 CD45CD10
CD9 CD63CD81
CD73 HLA-ABCCD29
CD44 CD90 CD105
% o
f Max
% o
f Max
A
Donor 1 Donor 2 Donor 30
500
1000
1500
2000
2500
3000N
umbe
r of e
xoso
mes
per
cel
l per
hou
r
*
Day 0 Day 1 Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Preparation
of bioreactor
Seeding
of
MSCs
Expansion of MSCs in growth media Wash with PBS and
load PLT-free media
A Timeline for MSCs-derived exosomes production using a bioreactor
Storage of MSCs iExo
aliquots at –80∘C
Thaw/Pool of
conditioned
media
Filtration
(0.2 µm)
Resuspension of MSCs-
derived exosomes in
research buffer (RB) or
clinical buffer (CB)
Ultracentrifugation
100,000g for 3 h
Electroporation
of exosomes
with KrasG12D
siRNA (iExo)
B Isolation of and generation of MSCs-derived iExosomes
C Generation of GMP grade MSCs-derived iExosomes
Day 11 Day 13 Day 15 Day 17 Day 19 Day 21
Bioreactor
Harvest 1
↓
Freeze (-80∘C)
Bioreactor
Harvest 2
↓
Freeze (-80∘C)
Bioreactor
Harvest 3
↓
Freeze (-80∘C)
Bioreactor
Harvest 4
↓
Freeze (-80∘C)
Bioreactor
Harvest 5
↓
Freeze (-80∘C)
Bioreactor
Harvest 6
↓
Freeze (-80∘C)
Supplemental Figure 4
Characterization of
exosomes by NanoSightTM
(30nm-250nm) and surface
markers (CD9, CD47,
CD29, CD105) + sterility
test
Dose preparations of
siRNA MSCs
exosomes for
infusion into patients
Day 0-19 Day 23-25
Pool of MSC-
conditioned
media and
centrifugation 1,000gfor 15 min at 4∘C
High speed
centrifugation
100,000g
Resuspension
of exosomes
+Exosomes siRNA
Day 40
Exosomes
carrying siRNA
Growth of MSC and
collection of
conditioned media
from the bioreactor
0.2 µm filter
Exosomes
pellet
Exosomes
pellet
(Sterility test from
each harvests)
Bioreactor
(Terumo BCT)
Cobe 2991 Cell
Processor
(Terumo BCT)
Bioreactor harvests:
Harvest time(days)
Volume(ml)
Protein (mg)
Exosomes (x109)
Harvest 1 10 250 2.641 2010
Harvest 2 12 250 6.222 4493
Harvest 3 14 250 3.606 3402
Harvest 4 16 250 3.534 3472
Harvest 5 18 250 0.992 983
Harvest 6 20 250 1.040 1210
C
0 5 10 15 2002468
10
80
100
120
140
Days
Leve
ls o
f glu
cose
and
lact
ose
Glucose (mg/dL)Lactose (mmol/L)
1 2 30
5
10
15
20
Num
ber o
f exo
som
es (1
012 )
Bioreactor run
BA
D
Harvest 1 Harvest 2
Harvest 3 Harvest 4
Harvest 5 Harvest 6
78.1% 85.0%84.6% 99.3%
97.2% 98.3%99.4%
92.9% 97.3%82.0% 80.0%
81.7%
93.2%
CD9 CD63 CD81 CD47
CD9 CD63 CD81 CD47
CD9 CD63 CD81 CD47
99.3%
99.0%
100% 100% 99.9%
100% 100% 99.9%
99.9% 95.3%
CD9 CD63 CD81 CD47
CD9 CD63 CD81 CD47
CD9 CD63 CD81 CD47
% o
f Max
% o
f Max
% o
f Max
97.1%
Supplemental Figure 5
0 10 20 30 40 50 600
1×107
2×107
3×107
4×107
Days (Post tumor induction)
Tota
l flux
(p/s
ec)
Control ExoBJ siKrasG12D-1 iExoMSCsiKrasG12D-1 iExo
Treatment Start
Supplemental Figure 6P
an
cre
as
Liv
er
Sp
lee
nK
idn
ey
Control Exo MSCs siKrasG12D-1 iExoBJ siKrasG12D-1 iExo
Contro
l Exo
BJ siK
rasG
12D-1 iE
xo
MSCs siK
rasG
12D-1 iE
xo0
5
10
15
Tum
or w
eigh
t at
ex
perim
enta
l en
dpoi
nt (g
)
Contro
l Exo
BJ siK
rasG
12D-1 iE
xo
MSCs siK
rasG
12D-1 iE
xo0
10
20
30
40
Tum
or b
urde
n at
ex
perim
enta
l en
dpoi
nt (%
)
E F
B
C
G
D
0
25
50
75
100
% o
f mic
e
Macrometastases/local invasiion detectedNo macrometastasis/local invasion detected
splee
nlun
gliv
er
Control Exo (n=4)
MSC siKrasG12D-1 iExo (n=6)
diaph
ragmkid
ney
splee
nlun
gliv
er
diaph
ragmkid
ney
splee
nlun
gliv
er
diaph
ragmkid
ney
BJ siKrasG12D-1 iExo (n=6)
Survival (Days) Pre-treatment End-point Observations Notes
Treatment PTS PB BW(g) TW(g) BW(g) Lung macro metastases Necropsy notes Suspected tumor
burden related deathMSCs Ctrl Exo 44 107 20.50 4.81 23.11 59
Large primary and secondary tumors, spleen invasion, liver invasion, diapragm invasion, macroscopic lung nodules
Yes
MSCs Ctrl Exo 37 100 20.00 7.00 23.90 49Large primary and secondary tumors, spleen invasion, diapragm
invasion, kidney invasion, macroscopic lung nodulesYes
MSCs Ctrl Exo 37 100 21.30 5.34 24.12 42Large primary and secondary tumors, diapragm invasion, macroscopic
lung nodulesYes
MSCs Ctrl Exo 44 107 21.60 6.22 27.26 76Large primary and secondary tumors, spleen invasion, liver invasion,
diapragm invasion, macroscopic lung nodulesYes
Average: 5.84BJ siKrasG12D-1 iExo 37 100 20.80 2.14 23.20 35 Moderate tumor, spleen invasion, macroscopic lung nodules Yes
BJ siKrasG12D-1 iExo 49 112 20.20 3.18 25.89 0 Moderate tumor, hunched, weak Yes
BJ siKrasG12D-1 iExo 66 129 20.50 7.92 31.24 0 Moderate tumor burden, hunched, spleen invasion, liver invasion Yes
BJ siKrasG12D-1 iExo 75 138 19.70 2.41 22.87 2Moderate tumor burden, spleen invasion, necrosis of primary tumor,
macroscopic lung nodulesYes
BJ siKrasG12D-1 iExo 101 164 20.30 6.40 28.31 0 Moderate tumor burden, hole in primary tumor hunched, weak Yes
BJ siKrasG12D-1 iExo 120 183 19.80 5.43 32.64 5 Moderate tumor burden, macroscopic lung nodules Yes
Average: 4.58MSCs siKrasG12D-1 iExo 54 117 21.30 1.62 26.40 0 Small tumor, no invasion No
MSCs siKrasG12D-1 iExo 54 117 21.60 2.76 31.86 0 Small tumor, no invasion, some internal bleeding Yes
MSCs siKrasG12D-1 iExo 75 138 19.70 5.64 23.00 0 Moderate tumor burden, no invasion, necrosis of primary tumor Yes
MSCs siKrasG12D-1 iExo 78 141 19.80 2.46 22.93 0 Moderate tumor, tumour in thoracic cavity, necrosis of primary tumor Yes
MSCs siKrasG12D-1 iExo 82 145 21.00 4.01 33.46 6Moderate tumor, necrosis of primary and secondary (subcutaneal) tumors, spleen invasion, kidney invasion, macroscopic lung nodules
Yes
MSCs siKrasG12D-1 iExo 115 178 21.00 11.00 35.48 32Moderate tumor burden, spleen invasion, kidney invasion, diagraphm
invasion, macroscopic lung nodulesYes
Average: 4.58
A
Untrea
ted
Contro
l Exo
MSC siKra
sG12
D-1 iExo
0
10
20
30
40
50P
erce
ntag
e of
apo
ptot
ic c
ells **
Figure 6BUntreated LS HS
13.2 78.0 86.312.9 90.0 89.011.1 84.0 82.0
Figure 6C
Untreated 11.19 8.69 9.48 1.00 1.00 1.00LS 13.28 11.10 12.76 0.23 0.19 0.10HS 13.20 12.95 13.50 0.25 0.05 0.06
Figure 6DsiKrasG12D-1 input siKrasG12D-2 input MSC siKrasG12D-1 iExo, HS, CB MSC siKrasG12D-1 iExo, HS, CB + RN MSC siKrasG12D-1 iExo, HS, CB + T + RN MSC siKrasG12D-1 iExo, LS, RB BJ siKrasG12D-1 iExo, LS, RB MSC siKrasG12D-2 iExo, LS, RB
0.05304851 0.05296884 0.05365822 0.05240104 0.04080845 0.05203353 0.05106859 0.051063970.05272926 0.05380001 0.03918899 0.05270242 0.05184339 0.051577130.05333581 0.05158886 0.04099962 0.05054021 0.05079002 0.05264135
Figure 6E, F: see Supplemental Figures 8C and 9B, respectively.
Delta Ct fold
Control Exo
BJ siKrasG12D iExo
Research Buffer (RB)
MSC siKrasG12D iExo
Research Buffer (RB)
MSCs siKrasG12D iExo
Clinical Buffer (CB)
Supplemental Figure 7
EB
F
Day 3
5D
ay 8
4
Control
Exo
BJ iExo
RB
MSCs iExo
RB
MSCs iExo
CB
0 20 40 60 80 100 120 140 160 1800.0
5.0×105
1.0×106
1.5×106
2.0×106
2.5×106
3.0×106
Days (Post tumor induction)
Rad
ianc
e (p
/sec
/cm
2 /sr
)
Control ExoBJ iExo RBMSCs IExo RBMSCs iExo CB
Start Treatment
Stop Treatment
C D
A
3 out 5
mice alive
4 out 4
mice alive5 out 5
mice alive
Contro
l Exo
BJ iExo
RB
MSCs iExo
RB
MSCs iExo
CB
0
2
4
6
8
Tum
or w
eigh
t at
expe
rimen
tal
endp
oint
(g)
Contro
l Exo
BJ iExo
RB
MSCs iExo
RB
MSCs iExo
CB
0
5
10
15
20
Tum
or b
urde
n at
ex
perim
enta
l en
dpoi
nt (%
)B
J iE
xo
MS
Cs
iExo
C
ontro
l Exo
Unt
reat
ed B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
B14
B15
B16
0
20
40
60
80
100
Per
cent
age
of a
popt
otic
cel
ls
BJ
iExo
M
SC
s iE
xo
Con
trol E
xoU
ntre
ated C
1C
2C
3C
4C
5C
6C
7C
8C
9C
10C
11C
12C
13C
14C
15C
16
0
20
40
60
80
100
Per
cent
age
of a
popt
otic
cel
ls
BJ
iExo
M
SC
s iE
xo
Con
trol
Exo
Unt
reat
ed A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
0
20
40
60
80
100
Per
cent
age
of a
popt
otic
cel
lsG
Survival (Days) Pre-treatment End-point ObservationsTreatment PTS PB BW(g) TW(g) BW(g) Necropsy notes
MSCs Ctrl Exo 70 133 29.40 1.89 23.00 Small tumor, hunched, diarrhea, no invasion
MSCs Ctrl Exo 71 134 31.10 2.96 33.26 Moderate tumor burden, spleen and liver invasion, ascistes
MSCs Ctrl Exo 85 148 28.70 2.03 30.57 Moderate tumor burden, diaphragm, spleen and kidney invasion
MSCs Ctrl Exo 99 162 34.90 3.26 36.22Moderate tumor burden, abdominal inflammation, ascistes, spleen
and diaphragm invasion
MSCs Ctrl Exo 113 176 25.50 3.10 28.97 Moderate tumor bourden, diaphragm invasion, ascistis
Average: 2.65BJ siKrasG12D-1 iExo RB 113 176 29.30 0.30 25.00 No tumor, hunched posture, diarrhea, no invasion
BJ siKrasG12D-1 iExo RB 143 206 28.20 2.83 27.21 Moderate tumor burden, hunched posture weak, no invasion
BJ siKrasG12D-1 iExo RB 145 208 29.00 4.05 38.50 Moderate tumor burden, hepatomegaly and splenomegaly
BJ siKrasG12D-1 iExo RB 165 228 27.80 2.36 28.00 Moderate tumor burden, spleen invasion
BJ siKrasG12D-1 iExo RB 221 284 31.80 2.54 28.20 Moderate tumor burden,diaprahm, liver and spleen invasion
Average: 2.42
MSCs siKrasG12D-1 iExo RB 165 228 31.30 1.47 21.00Severe abdominal inflammation, diapragm and kidney invasion,
mesenteric metastases
MSCs siKrasG12D-1 iExo RB 145 208 33.00 6.27 36.00 Large primary tumor, diaphragm, liver and spleen invasion
MSCs siKrasG12D-1 iExo RB 130 193 32.00 3.39 28.25 Moderate tumor burden, no invasion
MSCs siKrasG12D-1 iExo RB 198 261 27.90 0.5 25.00 Small tumor burden, no invasion
Average: 2.91BJ siKrasG12D-1 iExo CB 111 174 31.50 1.15 21.30 Moderate tumor burden, diaphragm invasion
BJ siKrasG12D-1 iExo CB 117 180 27.40 1.24 34.40 Moderate tumor, no invasion, ascistes
BJ siKrasG12D-1 iExo CB 221 284 30.40 0.31 18.00No tumor, hunched posture, weak, diarrhea, severe weight loss, no
invasion
BJ siKrasG12D-1 iExo CB 226 289 28.60 2.29 32.71 Moderate tumor burden, diaphragm and, liver invasion
BJ siKrasG12D-1 iExo CB 335 398 27.50 2.27** 30.59
Mouse did not reach moribundancy but showed decreased activity.
The pancreas appears normal yet a mesentaric tumor is noted
together with invasion in the kidneys.
Average: 1.25
Contro
l Exo
BJ siK
rasG
12D-1 iE
xo
MSC siKra
sG12
D-1 iExo
Contro
l Exo
PBS/Plas
malyte
Gemcit
abine
MSC siKra
sG12
D-2 iExo
Gemcit
abine
+MSC si
KrasG
12D-2 iE
xo0
2×106
4×106
6×106
8×106
1×107
Tota
l flux
(p/s
ec)
KPC689Experiment-1
KPC689Experiment-2
* Supplemental Figure 8
PBS/CB buffer MSC siKrasG12D-2 iExo Control Exo GemcitabineGemcitabine +
MSC siKrasG12D-2 iExo D
B
C
0 29 50 76
Time (days)
Tumor implantation
Treatment windows
Gem or PBS
Control Exo or iExo
A
Survival (Days) Pre-treatment End-point Observations
Treatment PTS PB BW(g) TW(g) BW(g) Lung Metastases Necropsy notes
CB/PBS 32 95 21.20 3.26 24.00 0 Moderate tumor burden, diaphragm invasionCB/PBS 34 97 20.50 4.90 21.97 4 Moderate tumor burden, spleen and diaphragm invasion, macroscopic lung nodules
CB/PBS 35 98 19.30 2.55 18.22 0 Moderate tumor burden, peritoneal, diaphragm, spleen iand kidney invasion, macroscopic lung nodules
CB/PBS 35 98 20.90 2.86 20.40 0 Moderate tumor burden, diaphragm invasionCB/PBS 41 104 20.78 2.14 20.78 6 Moderate tumor burden, peritoneal and diaphragm invasion, macroscopic lung nodulesCB/PBS 39 102 19.70 3.75 18.85 0 Moderate tumor burden, spleen, liver and kidney invasionCB/PBS 41 104 21.50 3.26 24.02 0 Moderate tumor burden, diaphragm invasionAverage: 3.25
MSCs Ctrl Exo 42 105 21.10 3.72 20.20 0 Moderate tumor burden, spleen, diaprahm invasion
MSCs Ctrl Exo 39 102 19.80 5.10 23.60 8 Moderate tumor burden, spleen, kidney, liver and diaprahm invasion, macroscopic lung nodules
MSCs Ctrl Exo 50 113 18.50 6.32 24.98 >200 Moderate tumor burden, spleen, kidney and diaprahm invasion, macroscopic lung nodules
MSCs Ctrl Exo 46 109 19.30 3.67 19.70 2 Moderate tumor burden, spleen and kidney invasion, macroscopic lung nodulesMSCs Ctrl Exo 34 97 20.90 2.86 17.50 0 Moderate tumor burden, spleen, liver and diaphragm invasionMSCs Ctrl Exo 36 99 21.30 2.83 22.80 0 Moderate tumor burden, spleen and diaphragm invasionMSCs Ctrl Exo 32 95 18.40 4.52 19.70 2 Moderate tumor burden, diaphragm invasion, macroscopic lung nodulesAverage: 4.15
Gemcitabine 39 102 18.70 0.54 23.47 0 Very small tumor, diaphragm invasion
Gemcitabine 41 104 20.20 2.54 24.66 1 Moderate tumor burden, spleen, kidney and diaphragm invasion, macroscopic lung nodules
Gemcitabine 50 113 21.90 2.3 18.00 0 Moderate tumor burden, no invasionGemcitabine 44 107 22.20 4.77 28.05 0 Moderate tumor burden, spleen, kidney and diaphragm invasion, ascitesGemcitabine 60 123 20.20 0.57 13.25 0 Small tumor, diaphragm invasionGemcitabine 46 109 19.10 1.5 27.20 0 Moderate tumor burden, no invasionGemcitabine 60 123 20.90 2.73 20.07 0 Moderate tumor burden, no invasionGemcitabine 57 120 20.40 3.1 22.90 0 Moderate tumor burden, no invasionAverage: 2.26
MSCs siKrasG12D-2 iExo CB 75 138 19.70 5.83 24.26 11 Moderate tumor burden, liver invasion, macroscopic lung nodulesMSCs siKrasG12D-2 iExo CB 45 108 22.40 1.96 20.00 13 Moderate tumor burden, spleen and diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB 45 108 18.80 1.78 16.15 29 Moderate tumor burden, spleen, kidney and diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB 44 107 20.60 1.72 18.87 0 Moderate tumor burden, spleen, kidney, peritoneal and diaphragm invasion
MSCs siKrasG12D-2 iExo CB 76 139 21.70 5.3 23.67 48 Moderate tumor burden, spleen, kidney and small intestine invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB 44 107 21.30 1.98 24.97 0 Moderate tumor burden, spleen, kidney, liver and diaphragm invasionMSCs siKrasG12D-2 iExo CB 36 99 21.00 1.34 21.60 0 Moderate tumor burden, diaphragm invasionMSCs siKrasG12D-2 iExo CB 44 107 18.90 1.86 20.00 15 Small tumor, diaphragm invasion, lymph node metastases, macroscopic lung nodules
Average: 2.72MSCs siKrasG12D-2 iExo CB+Gem 67 130 20.20 3.32 23.84 >200 Moderate tumor burden, spleen and kidney invasion, macroscopic lung nodulesMSCs siKrasG12D-2 iExo CB+Gem 46 109 20.60 1.14 19.85 0 Small tumor, hunched posture, liver invasion
MSCs siKrasG12D-2 iExo CB+Gem 75 138 19.20 2.27 21.00 8 Moderate tumor burden, spleen, kidney, liver and diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB+Gem 70 133 19.20 3.87 23.84 11 Moderate tumor burden, spleen, kidney and diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB+Gem 50 113 21.00 4.56 23.00 71 Moderate tumor burden, ascites, spleen, kidney and diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB+Gem 50 113 18.40 1.73 19.84 0 No tumor, hunched posture, weak, diarrhea, weight loss, no invasion
MSCs siKrasG12D-2 iExo CB+Gem 95 158 20.20 3.10 25.00 10 Moderate tumor burden, spleen, liver and diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB+Gem 87 150 20.00 1.46 16.44 0 Moderate tumor burden, spleen, liver and diaphragm invasionAverage: 2.68
Supplemental Figure 9
0 18 37 89
Time (days)
Tumor implantation
Treatment windows
Gem or PBS
Control Exo or iExo
A
B Survival (Days) Pre-treatment End-point Observations
Treatment PTS PB BW(g) TW(g) BW(g) Lung Metastases Necropsy notes
CB 46 116 19.50 4.14 25.50 6Large primary tumor, secondary tumor, peritoneal, diaphragm, stomach, spleen and liver invasion, mesenteric lymph node metastasis,
macroscopic lung nodules
CB 37 107 19.00 5.04 24.95 >100 Moderate tumor, spleen, liver, diaphragm and stomach invasion, macroscopic lung nodules
CB 47 117 20.00 1.36 16.90 0 Small tumor, bleeding, diaphragm invasion, mesenteric lymph node metastasis
CB 37 107 20.00 5.35 22.56 34 Moderate tumor, paraaortic lymph node metastasis, diaphragm and spleen invasion, possible stomach invasion, macroscopic lung nodules.
CB 42 113 19.50 1.80 25.00 14Moderate tumor, bleeding, mesenteric lymnph node metastases, diaphragm invasion, liver invasion, spleen invasion, macroscopic lung
nodules.
CB 37 107 19.00 2.73 18.71 9 Moderate tumor, spleen, lymph node and diaphragm invasion, macroscopic lung nodules
CB 53 124 20.00 3.46 26.04 3Large primary tumor, secondary tumor, peritoneal, diaphragm, spleen and liver invasion, mesenteric lymph node metastases, macroscopic
lung nodules
Average: 3.41
MSCs Ctrl Exo 42 113 19.00 5.43 29.60 9Large primary tumor, bleeding, ascites, peritoneal, diaphragm, stomach and kidney invasion, lymph node metastases, macroscopic lung
nodules
MSCs Ctrl Exo 42 113 20.00 1.63 22.00 2 Moderate tumor, diaphragm, spleen, peritoneal and lymph node invasion, macroscopic lung nodules
MSCs Ctrl Exo 60 131 19.5 7.12 27.8 >100Large primary tumor, bleeding, ascites, liver, diaphragm, stomach, ovary and spleen invasion, mesenteric lymph noode metastases,
macroscopic lung nodules
MSCs Ctrl Exo 39 109 20.00 2.02 22.16 >100Moderate tumor, diaphragm invasion, liver invasion, spleen invasion, peritoneal invasion, Mesenteric lymph node invasion, macroscopic lung
nodules
MSCs Ctrl Exo 41 112 19.50 4.18 23.37 32 Large primary tumor, bleeding, diaphragm invasion, stomach invasion, kidney invasion, spleen invasion, macroscopic lung nodules
MSCs Ctrl Exo 52 123 20.00 3.72 25.18 3 Large primary tumor, ascites, bleeding, diaphragm, stomach, liver, kidney and spleen invasion, lymph node metastases, paraaortic lymph node metastases, hydronephrosis, ovarian metastases, macroscopic lung nodules
MSCs Ctrl Exo 39 109 20.00 2.55 22.50 16Moderate tumor, bleeding, ascites, diaphragm, stomach, liver, spleen and peritoneal invasion, mesentereic lymph node invasion,
macroscopic lung nodules
Average: 3.81
Gemcitabine 54 125 20.00 3.08 25.96 19Large primary tumor, bleeding, ascites, spleen, liver, stomach, diaphragm, peritoneal, ovarian and left kidney invasion, pleural bleeding,
macroscopic lung nodules
Gemcitabine 56 127 20.00 3.49 25.38 >100Large primary tumor, bleeding, ascites, diaphragm, stomach, ovary, spleen and small instestine invasion, posible liver invasion,
macroscopic lung nodules
Gemcitabine 67 138 21.00 2.94 26.80 7Moderate tumor divided in two masses connected with necrotic core, very discolored liver (potential metastases), ovary and diaphragm
invasion, macroscopic lung nodules.
Gemcitabine 89** 160 20.00 1.03 21.90 0 Ascites, small tumor, possible spleen invasion and inflammation, necrotic liver
Gemcitabine 54 125 19.50 2.46 22.47 33Moderate tumor, bleeding, ascites, spleen, ovary and diaphragm invasion, hepatic lymph node metastases, paraaortic mesenteric lymph
node metastases, macroscopic lung nodules
Gemcitabine 67 138 20.00 2.49 27.70 2 Moderate tumor divided in two mass connected, very discolored liver (potential metastases), macroscopic lung nodules
Gemcitabine 89** 160 19.50 1.63 25.60 0Moderate tumor, bleeding, ascites, primaric tumor attached to the abdominal wall and connected to the pancreas. Possible spleen invasion
and inflammation, peripancreatic lymph node metastases, diaphragm invasion
Average: 2.45MSCs siKrasG12D-2 iExo CB 89** 160 20.00 2.58 23.60 0 Amall primary tumor, some ascites, peritoneal secondary tumor, diaprahgm invasion
MSCs siKrasG12D-2 iExo CB 66 137 20.00 4.18 25.58 5Large primary tumor with necrotic core, small secondary tumors (2), bleeding, ascites, lymph node metastases, spleen, liver, stomach and
kidney invasion, diaphragm metastases, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB 43 114 20.00 0.81 16.81 0 Moderate tumor, tumor with necrotic core (hole), bleeding, ascites, diaphragm invasion
MSCs siKrasG12D-2 iExo CB 56 127 20.00 5.06 25.90 34Large primary tumor, tumor with necrotic core (hole), bleeding, ascites, mesenteric lymph node and colon invasion, macroscopic lung
nodules
MSCs siKrasG12D-2 iExo CB 89** 160 20.00 2.4 16.00 29Moderate primary tumor, secondary tumor not connected to the primary tumor, tumor with necrotic core (hole), diapragm invasion,
macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB 67 138 20.00 7.7 30.21 2 Large primary tumor, peritoneal lymph node and diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB 89** 160 20.00 0.2 23.40 0 Remnant of primary tumor, some ascites, no invasion
Average: 3.28MSCs siKrasG12D-2 iExo CB+Gem 89** 160 20.00 1.80 23.10 0 Small primary tumor, no invasion
MSCs siKrasG12D-2 iExo CB+Gem 89** 160 19.50 0.20 20.10 0 Very small, remnant of tumor, large area of normal pancreas, edema in the capsule, no invasion
MSCs siKrasG12D-2 iExo CB+Gem 89** 160 20.00 2.24 23.30 0 Small primary tumor, area of normal pancreas, edema around the capsule, no invasion
MSCs siKrasG12D-2 iExo CB+Gem 89** 160 20.00 1.70 23.10 7Some ascites, spleen and diaphragm invasion, possible liver invasion, mesenteric lymph node metastases, lymph node metastases around
the stomach, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB+Gem 59 130 20.00 1.52 22.56 20 Small primary tumor, diaphragm invasion, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB+Gem 65 136 20.00 3.25 30.88 9Small primary tumor, bleeding, ascites, spleen, stomach, kidney, ovary and diaphragm invasion, hepatic lymph node metastases, lymph
node metastases, macroscopic lung nodules
MSCs siKrasG12D-2 iExo CB+Gem 89** 160 20.00 3.68 25.00 18Large primary tumor with area of normal pancreas, some ascites, bleeding, subcutaneous edema in the left flank, diaphragm invasion,
macroscopic lung nodules
Average: 2.06
A
PBS BJ Exo2.0
2.5
3.0
3.5
4.0
4.5A
lbum
in g
/dL
PBS BJ Exo0
50
100
150
Alk
alin
e P
ho
sph
ata
se U
/L
PBS BJ Exo0
50
100
150
200
250
ALT
U/L
PBS BJ Exo0
100
200
300
AS
T U
/L
PBS BJ Exo0
10
20
30
40
BU
N m
g/dL
PBS BJ Exo
1.6
2.0
2.4
Glo
bulin
g/d
L
PBS BJ Exo4
5
6
7
8T
otal
pro
tein
g/d
L
PBS BJ Exo0
5
10
15
Wh
ite b
loo
d c
ell
cou
nt 1
03/u
l
PBS BJ Exo7
8
9
10
11
12
Re
d b
loo
d c
ell
cou
nt 1
06 /
ul
Supplemental Figure 10
74K-Mesenteric lymph node-macrophages with yellow pigment
75K-Kidney inflammation, acute multifocal interstitial
75K-Liver inflammation-acute multifocal
74K-Liver hyperplasia bile ducts
69K-Normal Liver
72k-Inflammation gall bladder
69K Normal gall bladder
B
Spleen ThymusBone marrow
Supplemental Figure 11
A
B
CB liposomes BJ Exo MSC Exo MSC iExo0
5
10
15
20
IL-6
(pg
/mL)
0 0 0 0 0 0 0 0 00 0 0
CB liposomes BJ Exo MSC Exo MSC iExo20
25
30
35
40
% o
f T c
ells
(CD
3+ c
ells
)ou
t of t
otal
leuk
ocyt
es
CB liposomes BJ Exo MSC Exo MSC iExo12
14
16
18
20
22
24
% o
f CD
4+ T
cel
ls
out o
f tot
al le
ukoc
ytes
CB liposomes BJ Exo MSC Exo MSC iExo6
8
10
12
14
% o
f CD
8+ T
cel
ls
out o
f tot
al le
ukoc
ytes
CB liposomes BJ Exo MSC Exo MSC iExo0
5
10
15
% o
f Mye
loid
cel
ls (C
D11
b+ c
ells
) ou
t of t
otal
leuk
ocyt
es
CB liposomes BJ Exo MSC Exo MSC iExo45
50
55
60
65
70
% o
f CD
19+
B c
ells
out o
f tot
al le
ukoc
ytes
CB liposomes BJ Exo MSC Exo MSC iExo0
5
10
15
20
25
% o
f F4/
80+
cells
ou
t of t
otal
CD
11b+
cel
ls
CB liposomes BJ Exo MSC Exo MSC iExo0
2
4
6
8
% o
f T c
ells
(CD
3+ c
ells
)ou
t of t
otal
leuk
ocyt
esCB liposomes BJ Exo MSC Exo MSC iExo
0.0
0.5
1.0
1.5
2.0
2.5
% o
f CD
4+ T
cel
ls
out o
f tot
al le
ukoc
ytes
CB liposomes BJ Exo MSC Exo MSC iExo0.0
0.5
1.0
1.5
2.0
% o
f CD
8+ T
cel
ls
out o
f tot
al le
ukoc
ytes
CB liposomes BJ Exo MSC Exo MSC iExo50
60
70
80
90
% o
f Mye
loid
cel
ls (C
D11
b+ c
ells
) ou
t of t
otal
leuk
ocyt
es
CB liposomes BJ Exo MSC Exo MSC iExo0
5
10
15
20
% o
f CD
19+
B c
ells
ou
t of t
otal
leuk
ocyt
es
CB liposomes BJ Exo MSC Exo MSC iExo0
2
4
6
8
% o
f F4/
80+
cells
ou
t of t
otal
CD
11b+
cel
ls
CB liposomes BJ Exo MSC Exo MSC iExo70
75
80
85
90
95
% o
f T c
ells
(CD
3+ c
ells
)ou
t of t
otal
leuk
ocyt
es
#
CB liposomes BJ Exo MSC Exo MSC iExo0
2
4
6
8
% o
f CD
4+ T
cel
ls
out o
f tot
al le
ukoc
ytes
CB liposomes BJ Exo MSC Exo MSC iExo0.0
0.5
1.0
1.5
2.0
2.5
% o
f CD
8+ T
cel
ls
out o
f tot
al le
ukoc
ytes
‡
Lung
Kidney
Intestine
Spleen
Femur
Pancreas/Tumor
Liver
Lung
Kidney
Intestine
Testis
Femur
Liver
Spleen
Pancreas
Supplemental Figure 12A
Control DiR
Non tumor bearing Tumor bearing
Mouse 1 Mouse 2 Control DiR Mouse 1 Mouse 2
B
Kidney
Intestine
Femur
Lung
Brain
Heart
Kidney
Intestine
Femur
Lung
Brain
Heart
Pancreas
Spleen
Liver
Control DiR i.p. i.v.Control DiR i.p. i.v.
Radiant Efficiency
p/sec/cm2 /sr
!W/cm
2
0.5x108
1.5 x 108
Brain
Heart
Lung
Stomach
Bowel
Liver
Kidney
SpleenOvary
Femur
Control DiR 24hrs 48hrsControl DiRC
Radiant Efficiency p/sec/cm
2 /sr!W
/cm2
0.5 x108
2.5 x 108
2x108
Radiant Efficiency p/sec/cm
2 /sr!W
/cm2
6.0 x 108
Control DyR i.p. i.v.Pancreas
0.5 x108
2.5 x 108R
adiant Efficiency p/sec/cm
2 /sr!W
/cm2
1.0 x107
6.0 x 107
Radiant Efficiency
p/sec/cm2 /sr
!W/cm
2
1.0 x108
3.0 x 108
Supplemental Figure 13A
B
Untreated Control Exo
5.1%
8.7%
2%
8.3%
57%
21%
57%
30%
FreshFrozen (3 months)
/Thaw
Untreated Control Exo
FreshFrozen (6 months)
/Thaw
1%
4.3%
75.4%
7.5%
83%
3.4%
5.5%
3.1%
Annexin V Annexin V
Annexin V Annexin V
Annexin V Annexin V
Annexin V Annexin V
Mouse ID Treatment Age: Treatment start Survival:PB BW (g) TW (g)
sr31 siKrasG12D iExo 65 117 13.52 0.28sr96 siKrasG12D iExo 66 112 12.63 1.1sr23 siKrasG12D iExo 69 122 19.66 0.75
sm782 siKrasG12D iExo 75 140 8.17 0.14J44 siKrasG12D iExo 60 150 33.1 1.03J45 siKrasG12D iExo 60 98 16.8 1.44J43 siKrasG12D iExo 60 158 20.9 <0.1
Average 17.83 0.79sr29 Control Exo 64 110 18.73 0.3
sr257 Control Exo 58 92 18.2 1.28sr270 Control Exo 60 69sr271 Control Exo 60 85 14.45 0.94sr475 Control Exo 59 101 16.2 1.45J54 Control Exo 61 113 16.66 1.3
Average 16.85 1.05
End point
N/A
LIVE
/DEA
D™
Fixa
ble
Aqua
Sta
inLI
VE/D
EAD
™Fi
xabl
e Aq
ua S
tain
LIVE
/DEA
D™
Fixa
ble
Aqua
Sta
inLI
VE/D
EAD
™Fi
xabl
e Aq
ua S
tain
Treatment Group PBS PBS PBS Exo Exo Exo ExoAnimal ID 69K 70k 71K 72K 73K 74K 75KSpecie mouse mouse mouse mouse mouse mouse mouseStrain/Genotype C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6JTerminal body weight (grams) 37.16 35.04 40.28 35.81 32.52 27.84 29.76Liver weight (grams) 2.02 1.99 1.99 1.97 1.71 1.47 1.52Kidney weight (grams) - Right/Left 0.49 0.45 0.57 0.51 0.46 0.43 0.45Heart weight (grams) 0.17 0.21 0.25 0.15 0.16Spleen weight (grams) 0.08 0.07 0.11 0.08 0.07 0.10 0.10Block No.
OrganMorphologic Diagnosis
Lesion Grade
Lesion Grade
Lesion Grade
Lesion Grade
Lesion Grade
Lesion Grade
Lesion Grade
A Liver A A N N A A AInflammation, acute, mixed, multifocal 1 1 2Inflammation, acute, bile ducts 1 2Hyperplasia, bile ducts 1 1Extramedullary hematopoiesis, (incidental finding) 1 1 2
A Gall Bladder N N N A A A AInflammation, acute, neutrophilic 2 1 1Distended/dilated 3
A Kidneys A A A A A A AInflammation, acute, mixed (neutrophils and macrophages), interstitial, multifocal 2
Chronic Progressive Nephropathy (multifocal hyperplasia of cortical renal tubules; and/or proteinaceous tubular casts in the medullary renal tubules) (incidental age-related change)
1 1 1 1 1 2
Lymphoplasmacytic infiltration, perivascular, arcuate and mid-size blood vessels (incidental age-related change)
1 1 1 2 1 1 2
B Lung A N A N N A N
Inflammation, acute, mixed, perivascular 1 2 2Increased number of alveolar macrophages, diffuse 1
C Heart N N N N N N N
E Brain N A N N A N NMineralization/calcification, multifocal, neuropil, thalamus, (incidental finding)
1 1
M Mesenteric Lymph Node and Mesentery 0 A A N A A 0Hyperplasia, paracortex 2 2 2Hyperplasia, lymphoid follicles 2 1Apoptotic lymphocytes with tangible-body macrophages, paracortex
2 3 2 2
Macrophages with intracytoplasmic pale-yellow pigment/material in the medullary sinuses
1 2 3
Inflammation, acute (infiltration of neutrophils) 2Infiltration of mast cells in the medullary sinuses 2Plasmacytosis (increased number of plasma cells) 2
M Spleen N N A A A A AHyperplasia, lymphocytic, periarteriolar lymphoid sheaths (white pulp)
1 2 1
Plasmacytosis (increased number of plasma cells) 2 1 2 1 2T Bone marrow, femur, knee joint N N N N N N N
U Bone marrow, sternum N N N A N N NPlasmacytosis, focal 1
Comment:Liver: Minimal to mild acute inflammation around the bile ducts in mice #73K, 74K, 75K, and of gall bladder in mice #72K, 73K, 75K, indicate mild injury to cholangiolarepithelium of bile ducts and gall bladder. Mild multifocal acute inflammation of mononuclear cells and neutrophils in the liver of mouse #75K is consistent with mild injury/toxicity to hepatocytes. Kidney: Mild acute multifocal interstitial inflammation suggests mild toxicity to epithelium of renal tubules. Immune System: Minimal to mild plasmacytosis and lymphoid hyperplasia of spleen and mesenteric lymph nodes in mice 71K, 72K, 73K, 74K, 75K indicate mild immune activation of lymphoid system and increased production of antibodies by plasma cells. Presence of macrophages with intracytoplasmic pale-yellow pigment in the sinusoids of mesenteric lymph nodes indicates increased phagocytosis of either foreign material, red cells, lipofuscin or ceroid material. Additionally, the mesenteric lymph node of mouse 74K had mild acute inflammatory reaction suggesting a regional injury from gastrointestinal tract or peritoneum.
LEGENDN = Normal tissue or No significant lesion observedA = Abnormal tissue (Lesion present)0 = No tissue present on the slideP = The lesion is present, but not gradedS = Suspected/Presumptive diagnosis (needs more tests to confirm the Dx.)PMA = Postmortem autolysisI = Inadequate sectionN/A= Not ApplicableGRADING/SCORING OF HISTOLOGICAL LESIONSGrade 0 = no histologic lesion (or normal tissue)Grade 1 = minimal, minor, rare, infrequent, barely noticeable tissue change (lesion affects 1-10% of the tissue)Grade 2 = mild, slight, sporadic, noticeable but not prominent feature (lesion affects 11-20% of tissue)Grade 3 = moderate, frequent, typical, common, prominent tissue change (lesion affects 21-40% of tissue)Grade 4 = marked, extensive, numerous, severe, overwhelming tissue change (lesion affects 41-100% of tissue)
Supplemental Table 1