, 1612 (2011);
, et al. Gregory L. Beatty
Pancreatic Carcinoma in Mice and Humans
CD40 Agonists Alter Tumor Stroma and Show Efficacy Against
This copy is for your personal, non-commercial use only.
clicking here. colleagues, clients, or customers by
, you can order high-quality copies for your
If you wish to distribute this article to others
The following resources related to this article are available online at
here. following the guidelines
can be obtained by
Permission to republish or repurpose articles or portions of articles
Updated information and services,
): April 28, 2011 www.sciencemag.org (this infomation is current as of
version of this article at:
including high-resolution figures, can be found in the online
can be found at:
Supporting Online Material
related to this article
A list of selected additional articles on the Science Web sites
, 8 of which can be accessed free:
cites 22 articles
This article appears in the following
registered trademark of AAAS.
is a Science2011 by the American Association for the Advancement of Science; all rights reserved. The title
CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science
on April 28, 2011
ecosystem structure. Our study shows that de-
clines in salmon will have the largest ecological
impacts on smaller and less productive streams.
In contrast, large and shallow-sloped watersheds
dominated by nitrogen-fixing red alders are pre-
dicted to be more resilient to salmon declines.
These considerations enable predictions of im-
boundaries, which can then be incorporated into
References and Notes
1. B. Worm et al., Science 314, 787 (2006).
2. J. B. C. Jackson et al., Science 293, 629 (2001).
3. H. K. Lotze et al., Science 312, 1806 (2006).
4. G. A. Polis, S. D. Hurd, Am. Nat. 147, 396 (1996).
5. D. A. Croll, J. L. Maron, J. A. Estes, E. M. Danner,
G. V. Byrd, Science 307, 1959 (2005).
6. J. L. Maron et al., Ecol. Monogr. 76, 3 (2006).
7. L. B. Marczak, R. M. Thompson, J. S. Richardson, Ecology
88, 140 (2007).
8. S. M. Gende, R. T. Edwards, M. F. Willson, M. S. Wipfli,
Bioscience 52, 917 (2002).
9. R. J. Naiman, R. E. Bilby, D. E. Schindler, J. M. Helfield,
Ecosystems 5, 399 (2002).
10. D. J. Janetski, D. T. Chaloner, S. D. Tiegs, G. A. Lamberti,
Oecologia 159, 583 (2009).
11. J. W. Moore, D. E. Schindler, Can. J. Fish. Aquat. Sci. 61,
12. J. W. Moore et al., Ecology 88, 1278 (2007).
13. S. D. Tiegs et al., Ecol. Appl. 18, 4 (2008).
14. J. M. Helfield, R. J. Naiman, Oecologia 133, 573 (2002).
15. See supporting material on Science Online.
16. M. H. H. Price, C. T. Darimont, N. F. Temple,
S. M. MacDuffee, Can. J. Fish. Aquat. Sci. 65, 2712
17. T. E. Reimchen, Can. J. Zool. 78, 448 (2000).
18. C. T. Darimont, P. C. Paquet, T. E. Reimchen, BMC Ecol.
8, 14 (2008).
19. R. E. Bilby, B. R. Fransen, P. A. Bisson, Can. J. Fish.
Aquat. Sci. 53, 164 (1996).
20. K. K. Bartz, R. J. Naiman, Ecosystems 8, 529 (2005).
21. C. E. Wilkinson, M. D. Hocking, T. E. Reimchen, Oikos
108, 85 (2005).
22. M. D. Hocking, T. E. Reimchen, Oikos 118, 1307
23. K. Klinka, V. J. Krajina, A. Ceska, A. M. Scagel, Indicator
Plants of Coastal British Columbia (Univ. of British
Columbia Press, Vancouver, 1989).
24. J. M. Diez, H. R. Pulliam, Ecology 88, 3144 (2007).
25. A. F. Zuur, E. N. Ieno, N. J. Walker, A. A. Saveliev,
G. M. Smith, Mixed Effects Models and Extensions in
Ecology with R (Springer Science+Business Media, New
26. P. A. Alaback, Ecology 63, 1932 (1982).
27. J. Pojar, K. Klinka, D. V. Meidinger, For. Ecol. Manage.
22, 119 (1987).
28. G. Pinay, T. O'Keefe, R. Edwards, R. J. Naiman,
Ecosystems 6, 336 (2003).
29. E. A. Hobbie, J. E. Hobbie, Ecosystems 11, 815 (2008).
30. J. M. Craine et al., New Phytol. 183, 980 (2009).
31. J. M. Kranabetter, W. H. MacKenzie, Ecosystems 13,
32. J. J. Verspoor, D. C. Braun, J. D. Reynolds, Ecosystems 13,
33. R. E. Bilby, B. R. Fransen, J. K. Walter, C. J. Cederholm,
W. J. Scarlett, Fisheries 26, 6 (2001).
34. DFO (Fisheries and Oceans Canada, Vancouver, 2005).
35. C. T. Darimont et al., Conserv. Lett. 3, 379 (2010).
36. D. E. Schindler et al., Nature 465, 609 (2010).
37. Supported by the Tom Buell Endowment Fund at Simon
Fraser University (SFU), a Natural Science and
Engineering Research Council of Canada (NSERC)
postdoctoral fellowship (M.D.H.), NSERC Discovery and
Accelerator grants (J.D.R.), the B.C. Leading Edge
Endowment Fund, the Pacific Salmon Foundation, the
B.C. Pacific Salmon Forum, and Mountain Equipment
Co-op. We thank C. Aries for plant surveys; D. Braun,
A. Cooper, E. Darling, N. Dulvy, R. Field, J. Harding,
J. Linton, M. Stubbs, W. Palen, J. Verspoor, and the
Earth2Ocean Research Group at SFU for discussions
and analytical support; A. Albright, J. Barlow, J. Beaudin,
J. Gordon-Walker, I. Jansma, E. Nelson, M. Spoljaric,
J. Wilson, and the Raincoast Conservation Foundation for
field support; R. Carpenter, M. Reid, and others at the
Heiltsuk Integrated Resource Management Department;
L. Jorgenson from Qqs Projects Society in Bella Bella; and
the Heiltsuk Nation for research partnerships in their
Supporting Online Material
Materials and Methods
Figs. S1 to S5
Tables S1 to S13
30 November 2010; accepted 25 January 2011
CD40 Agonists Alter Tumor Stroma and
Show Efficacy Against Pancreatic
Carcinoma in Mice and Humans
Gregory L. Beatty,1,2,6Elena G. Chiorean,3Matthew P. Fishman,1Babak Saboury,5
Ursina R. Teitelbaum,2,6Weijing Sun,2,6Richard D. Huhn,4Wenru Song,4Dongguang Li,4
Leslie L. Sharp,4Drew A. Torigian,2,5Peter J. O’Dwyer,2,6Robert H. Vonderheide1,2,6*
Immunosuppressive tumor microenvironments can restrain antitumor immunity, particularly
in pancreatic ductal adenocarcinoma (PDA). Because CD40 activation can reverse immune
suppression and drive antitumor T cell responses, we tested the combination of an agonist CD40
antibody with gemcitabine chemotherapy in a small cohort of patients with surgically incurable
PDA and observed tumor regressions in some patients. We reproduced this treatment effect
in a genetically engineered mouse model of PDA and found unexpectedly that tumor regression
required macrophages but not T cells or gemcitabine. CD40-activated macrophages rapidly
infiltrated tumors, became tumoricidal, and facilitated the depletion of tumor stroma. Thus,
cancer immune surveillance does not necessarily depend on therapy-induced T cells; rather, our
findings demonstrate a CD40-dependent mechanism for targeting tumor stroma in the treatment
fit over best supportive care for patients who are
not surgical candidates (1). We have previously
demonstrated that leukocytes actively infiltrate
the stromal compartment of PDA, even at the
earliest stages of tumor development, and or-
chestrate an immune reaction that is immuno-
suppressive (2). In this study, we investigated
the reversibility of this immune suppression, hy-
ancreatic ductal adenocarcinoma (PDA)
remains an almost universally lethal dis-
ease; chemotherapy offers minimal bene-
pothesizing initially that doing so would unleash
tumor-specific cellular immunity to eliminate PDA.
Activation of the tumor necrosis factor (TNF)
receptor superfamily member CD40 has been
shown to be a key regulatory step in the devel-
opment of T cell–dependent antitumor immu-
nity, which relies on CD40-mediated “licensing”
of antigen-presenting cells (APCs) for tumor-
specific T cell priming and activation (3–8).
On the basis of this premise, we investigated
in both humans and mice whether systemic CD40
activation with an agonist CD40 monoclonal
antibody (mAb) can circumvent tumor-induced
immune suppression and invoke productive T
cell–dependent antitumor immunity in PDA.
We first evaluated the clinical impact of CD40
activation by performing a clinical trial of the fully
human agonist CD40 mAb CP-870,893 (9) in
combination with gemcitabine chemotherapy
(2′-deoxy-2′,2′-difluorocytidine) for patients with
chemotherapy-naïve, surgically incurable PDA
(10). We tested CP-870,893 with gemcitabine be-
cause chemotherapy delivered before an agonist
CD40 mAb can facilitate enhanced tumor anti-
gen presentation by APCs (11–14). Twenty-one
patients (90% with metastatic disease) received
gemcitabine weekly on days 1, 8, and 15 with
CP-870,893 administered on day 3 of each 28-
day cycle (fig. S1). Treatment was well tolerated
overall (fig. S2), and the most common side
effect was mild-to-moderate cytokine release
syndrome characterized by chills, fevers, rigors,
and other symptoms on the day of CP-870,893
1Abramson Family Cancer Research Institute, University of
Pennsylvania School of Medicine, 421 Curie Boulevard, Phil-
adelphia, PA 19104, USA.2Abramson Cancer Center, Univer-
sity of Pennsylvania School of Medicine, Philadelphia, PA
19104, USA.3Division of Hematology/Oncology, Department
of Medicine, Indiana University School of Medicine, Indian-
apolis, IN 46202, USA.4Pfizer Corporation, New London, CT
06320, USA.5Department of Radiology, Department of Med-
icine, University of Pennsylvania School of Medicine, Phila-
delphia, PA 19104, USA.6Division of Hematology-Oncology,
Department of Medicine, University of Pennsylvania School of
Medicine, Philadelphia, PA 19104, USA.
*To whom correspondence should be addressed. E-mail:
25 MARCH 2011 VOL 331
on April 28, 2011
infusion. Symptoms were transient (<24 hours)
and managed in the outpatient clinic with sup-
The impact of therapy on the primary and
metastatic lesions was evaluated using two
standard modalities: (i) [18F]2-fluoro-2-deoxy-
D-glucose (FDG) avidity on positron emission
tomography–computed tomography (PET-CT)
as a measure of an on-target biological effect
of therapy and (ii) CT imaging for Response
Evaluation Criteria in Solid Tumors (RECIST)
assessment. Metabolic responses were observed
by FDG–PET-CT in both the primary and meta-
static lesions in 88% of patients evaluated after
two cycles of therapy (fig. S3). By RECIST, 4
out of 21 patients developed a partial response
(PR), 11 patients had stable disease (SD), and
4 patients had progressive disease (PD) (Fig.
1A). Two patients were not evaluable on study
for tumor response. One of these patients (pa-
tient 10031010) was taken off the study proto-
col after one dose of CP-870,893 because of a
grade 4 cerebrovascular accident but recovered
neurologically and restarted gemcitabine alone
and achieved a PR. The second patient (patient
10031001) had clinical deterioration from dis-
ease progression, and posttherapy CT imaging
was not obtained. The median progression-free
survival (PFS) for the 21 patients was 5.6 months
(95% confidence interval, 4.0 months to not esti-
mable), and the median overall survival (OS)
was 7.4 months (95% confidence interval, 5.5
to 12.8 months). Median PFS and OS are based
on interim data as of 25 August 2010, with 6
of 21 patients alive. Gemcitabine alone achieves
a historical tumor response rate of 5.4%, with
median PFS of 2.3 months and median OS of
5.7 months (1). Therefore, treatment with CP-
870,893 and gemcitabine showed therapeutic
efficacy in patients with metastatic PDA.
One patient with a PR showed a 46% re-
duction in the primary pancreatic lesion, com-
plete resolution of a 7.6-cm hepatic metastasis,
and 47% reduction in the one remaining hepatic
metastasis (Fig. 1B). Upon biopsy after four
cycles of therapy, this remaining liver lesion
showed no viable tumor. Instead, we observed
necrosis and an infiltrate dominated by macro-
phages with an absence of lymphocytes (Fig.
1C). A second patient with a PR underwent
surgical resection of the primary tumor after
achieving a complete resolution of all hepatic
metastases and a 64% reduction in the primary
pancreatic lesion. Histological analysis of the
resected primary lesion revealed a cellular in-
filtrate devoid of lymphocytes (Fig. 1C). Such
tumor regression without lymphocyte infiltration
was unexpected on the basis of results from im-
plantable tumor models, where CD40-induced
antitumor activity is dependent on T cells (6–8).
Therefore, to understand the mechanism of CD40-
mediated tumor regression, we turned to a spon-
taneous mouse model of PDA.
The KPC model of PDA is a genetically
engineered mouse model that incorporates the
conditional expression of both mutant KrasG12D
and p53R172Halleles in pancreatic cells (15).
Because immunocompetent KPC mice sponta-
neously develop PDA tumors that display high
fidelity to the histopathologic and molecular fea-
tures of human PDA (15), the KPC model is
thought to be relevant for understanding treat-
ment mechanisms in patients (16, 17). We there-
fore modeled our clinical study in KPC mice by
evaluating the therapeutic efficacy of gemcita-
bine weekly with the agonist CD40 mAb FGK45
administered 48 hours after the first infusion of
gemcitabine. Tumor response was monitored by
three-dimensional ultrasonography (fig. S4) (10).
We found that the combination of FGK45 with
gemcitabine induced tumor regression in 30% of
mice (Fig. 2A), similar to the objective response
rate in our patients. Treatment with FGK45 alone
resulted in the same rate of tumor regression,
whereas gemcitabine alone did not (Fig. 2A).
Activation of the CD40 pathway is a critical
event in the development of tumor-specific T
cell immunity (18). To test whether CD40 im-
munotherapy is dependent on T cell activation,
we examined the function of T cells isolated
from the spleen and pancreas (including tumor
and peripancreatic lymph nodes) of KPC mice.
Seven days after treatment with FGK45, both
CD4+and CD8+T cells of KPC mice acquired
an increased capacity to secrete cytokines, in-
cluding interferon-g (IFN-g) and interleukin IL-
17A (fig. S5A); however, FGK45 induced the
same rate of tumor regression in KPC mice in
the absence of CD4+or CD8+cells or both CD4+
Fig. 1. Agonist CD40 mAb in combination with gemcitabine
induces clinical responses in patients with surgically incurable PDA.
(A) Best overall percentage of change from baseline in tumor target
lesion measurement shown as a waterfall plot. *Patient 10061001 was defined
as PD because of the appearance of a new nontarget lesion. **Patient 10031001
did not obtain posttherapy scans because of clinical deterioration from disease
progression after one dose of CP-870,893. ***Patient 10031010 came off the
study after one dose of CP-870,893 but restarted gemcitabine alone and achieved
a PR. (B) CT imaging obtained at baseline and end of cycle 3. The primary pan-
creatic tumor and two metastatic liver lesions are marked by arrows, with the
longest dimension annotated. (C) Histopathology of a biopsied metastatic lesion
[from patient 10031016 (left)] and a surgically resected primary pancreatic
lesion [from patient 10061003 (right)]. Both patients achieved a PR. Patient 10031016 underwent tumor biopsy after completing four cycles of therapy;
patient 10061003 underwent surgical resection of the primary tumor after 12 cycles of therapy. Arrows (left) indicate a macrophage-dominated inflammatory
infiltrate within extensive tumor necrosis. Arrows (right) identify polymorphonuclear infiltrating cells without lymphocytes; arrowheads mark tumor cells. Scale
bars, 50 mm.
VOL 33125 MARCH 2011
on April 28, 2011
and CD8+cells (Fig. 2A). This was unexpected
given the established link between CD40 ac-
tivation and the development of productive anti-
tumor T cell immunity (6–8). Histopathological
appearance of tumors from responder compared
with nonresponder animals at day 14 was sim-
ilar overall, with the exception that tumors were
smaller in responder animals (Fig. 2, B to E).
CD3+T cells were not observed to infiltrate tu-
mors in KPC mice at baseline or at any time
after treatment with FGK45 (Fig. 2, F to H).
Instead, CD3+T cells remained localized to
peripancreatic lymph nodes situated adjacent to
developing tumors (Fig. 2, E and I). Moreover,
when the whole pancreas was resected en bloc
with peripancreatic lymph nodes, CD3+T cells
did not change in frequency before and after
therapy (fig. S5, B and C). Thus, activation of
Fig. 2. Antitumor activity of
agonist CD40 mAb in KPC
mice is T cell–independent.
gemcitabine or phosphate-
or FGK45 administered on
ceiving treatment with FGK45
were also depleted of CD4+
or CD8+cells or both CD4+
and CD8+cells with the use
in tumor volume from day –1 (baseline) to day +14 is shown for each mouse as a
waterfall plot (in comparison with PBS + IgG2a, gemcitabine + FGK45: P < 0.05;
gemcitabine + IgG2a: P = 1.00; PBS + FGK45: P < 0.05; FGK45 + GK1.5: P < 0.05;
FGK + 2.43: P < 0.05; FGK45 + GK1.5 + 2.43: P < 0.05; Fisher’s exact test).
Hematoxylin and eosin (H&E) histology [(B) to (E)] and CD3 immunohistochemistry
(C to E and G to I). Responders (D), (E), (H), and (I) were defined as FGK45-treated
with tumor progression by ultrasound were defined as nonresponders (C) and (G).
Scale bars, 50 mm.
Fig. 3. Agonist CD40
mAb targets systemic
infiltration of tumor. Im-
pancreatic lymph nodes
mor (A, C, D, E, and G)
by staining with a bio-
tinylated goat antibody
directed against rat IgG
(A) and (E) to (G) after
were depleted with CELs
before FGK45 treatment
(A) and (G). LN, peripan-
mor; scale bars, 50 mm.
Error bars in (A) repre-
displaying the flow cy-
(columns) for expression
of cell surface molecules (rows) 3 days after treatment with control IgG2a
compared with FGK45. (Top) The percentage of tumor-associated macrophages
with surface molecule expression. (Bottom) Mean fluorescence intensity as the
number of standard deviations from the mean, which was determined from
treatment with control IgG2a. P values are based on Student’s t test; nd, not
25 MARCH 2011VOL 331
on April 28, 2011
the CD40 pathway does not trigger T cell infil-
tration into tumors and is insufficient to induce
productive antitumor Tcell immunity in the KPC
CD40 is expressed on a wide range of
leukocytes including monocytes, tissue macro-
phages, B cells, and dendritic cells and can also
be found on some tumors (19). By immuno-
histochemistry, we found that PDA cells were
CD40-negative, whereas cells in the tumor stroma
expressed CD40, particularly F4/80+tumor-
associated macrophages (fig. S6). We therefore
investigated the impact of CD40 immuno-
therapy on F4/80+macrophages in KPC mice.
Within 18 hours of administration, FGK45 in-
duced a transient depletion of macrophages from
the peripheral blood, with subsequent accumu-
lation in the spleen (fig. S7, A and B). To test
whether CD40 mAb initially engages CD40 on
peripheral blood macrophages, which then mi-
grate into tissues, we compared the biodistribu-
tion of FGK45 with that of RB6-8C5, a mAb
used as a control and specific for Gr-1 expressed
on granulocytes and immature myeloid cells in
peripheral blood (fig. S7, C and D). Within 2 to
10 min after systemic administration of RB6-
8C5, Gr-1+cells in the peripheral blood were
found to be coated with antibody (fig. S8A). In
contrast, FGK45 was not found on the cell sur-
face of F4/80+macrophages in peripheral blood
(fig. S8A) nor was FGK45 internalized after
binding to CD40 (fig. S8B). Instead, at 10 min,
FGK45 was observed bound to leukocytes re-
siding within the spleen, lymph nodes, and pan-
creas of KPC mice (fig. S9A). Within the tumor
microenvironment, FGK45 was found initially
to localize to peripancreatic lymph nodes (Fig.
3, A and B) and not the tumor (Fig. 3, A and C),
in contrast to RB6-8C5, which bound leuko-
cytes in the tumor periphery as early as 10 min
after treatment (Fig. 3D). At 18 hours, FGK45
was observed bound to F4/80+macrophages
infiltrating the tumor but not lymph nodes (Fig.
3, A, E, and F; and fig. S9, B and C). The
binding of FGK45 to leukocytes within the tumor
microenvironment was found to occur in clus-
ters of cells rather than diffusely. We observed
no change in the frequency of macrophages in
the pancreas after FGK45 treatment (fig. S10).
To determine whether FGK45 binds to macro-
phages before their infiltration of tumors, we
treated KPC mice with clodronate-encapsulated
liposomes (CELs) to deplete systemic macro-
phages (fig. S11A). Because liposomes are not
capable of traversing vascular barriers produced
by capillary walls, CELs do not diffuse into tis-
sues (20) and, therefore, do not deplete macro-
phages within tumors (fig. S11, B to E). When
KPC mice were treated with CELs, however,
FGK45 labeling was no longer detectable in the
tumors (Fig. 3, A and G), despite the continued
presence of CD40+macrophages within the tu-
mor. These findings support the hypothesis that
FGK45 binds to macrophages before their mi-
gration into tumors.
Depending on their phenotype, macrophages
can either promote or inhibit tumor progression
(21, 22). In the absence of any treatment, we
found that tumor-associated macrophages were
activated and capable of secreting IL-10, TNF-a,
and IL-6 but had reduced expression of MHC
class II molecules compared with splenic macro-
phages from tumor-bearing KPC mice and nor-
mal littermates (fig. S12). After treatment with
FGK45, macrophages in KPC tumors and spleens
Fig. 4. CD40 activated
rophages mediate tumor
regression. (A) KPC mice
were treated with control
plus depletion of systemic
macrophages by means
of CELs.Shown is awater-
fall plot displaying per-
cent change in tumor
volume from baseline to
day +14 (in comparison
with IgG2a, FGK45: P <
0.05; FGK45 + CELs: P =
1.00; Fisher’s exact test).
(B) F4/80+tumor-associated macrophages were isolated from
KPC mice that had been treated with FGK45 (blue squares)
or control IgG2a (green circles) and incubated with KPC-
derived tumor cell lines. Tumor cell death in vitro was
measured by 7-aminoactinomycin D (7-AAD) labeling and
flow cytometric analysis at 24 hours. Shown is a representative assay from
two independent experiments each performed in triplicate. Means T SD are
depicted; *P < 0.05, Student’s t test. (C) Cleaved caspase 3 expression on
KPC tumors was determined by immunohistochemistry 18 hours after
treatment with control IgG2a (top panel) or FGK45 (bottom panel). (D) to (L)
KPC tumors were analyzed 18 hours after treatment with control IgG2a (D,
F, and H), FGK45 (E, G, and I), or FGK45 + CEL (J, K, and L). Shown are
hematoxylin-and-eosin histology (D, E, J); Masson’s trichrome stain to reveal
extracellular matrix in blue [(F), (G), and (K)], and immunohistochemistry for
collagen I [(H), (I), and (L)]. Scale bars, 50 mm.
VOL 331 25 MARCH 2011
on April 28, 2011
in tumor-bearing KPC mice up-regulated MHC Download full-text
class II and the costimulatory molecule CD86
with expression levels peaking at day 3 and re-
turning to baseline after 7 days (Fig. 3H, and figs.
S13 and S14). These changes coincided with a
cytokine surge on day 1 after FGK45 treatment,
with elevated serum levels of IL-12, TNF-a, and
IFN-g, but not IL-10, in KPC animals (fig. S15).
To determine whether macrophages were
necessary for CD40-mediated tumor regression,
we depleted systemic macrophages from KPC
mice with CELs. Treatment with CELs abolished
the capacity of FGK45 to induce tumor regres-
sion (Fig. 4A). Macrophages isolated from the
pancreas of tumor-bearing KPC animals treated
in vivo with FGK45 lysed tumor cells in vitro
(Fig. 4B). This finding correlated with in vivo
observations of cleaved caspase 3 expression in
focal areas of the tumor at 18 hours after treat-
ment with FGK45 (Fig. 4C). At this time after
treatment, regions of the tumor stroma and as-
sociated fibrosis appeared to be undergoing
involution (Fig. 4, D to G). These regions dis-
played a decrease in collagen I content, consist-
ent with degradation of the tumor matrix (Fig.
4, H and I). In KPC mice depleted of systemic
macrophages using CELs, FGK45 treatment failed
to induce stromal degradation (Fig. 4, J to L).
These findings identify a novel mechanism where-
by the CD40 pathway can be harnessed thera-
peutically to restore tumor immune surveillance
by targeting tumor-infiltrating macrophages in-
volved in cancer inflammation.
PDA is a common, devastating, and highly
lethal tumor for which new therapies are crit-
ically needed. Our findings identify a previously
unappreciated role for the CD40 pathway in
regulating the immune reaction and fibrosis
associated with PDA by reeducation of tumor-
associated macrophages. Mechanistically, CD40
agonists altered tumor stroma and, in both mice
and humans, showed efficacy against PDA. Al-
though tumor-suppressing macrophages have
been previously described (22), their role has
been largely linked to the orchestration of T cell
antitumor immunity. In this study, CD40 acti-
vation was, by itself, insufficient for invoking
productive antitumor T cell immunity, and we
hypothesize that full engagement of T cell im-
munity in PDA after CD40 activation will re-
quire modulation of additional tumor and host
factors or the incorporation of novel vaccines
(23). Our results emphasize that tumor immu-
nosurveillance can at times be governed strictly
by innate immunity under the regulation of the
CD40 pathway and support the continued de-
velopment of emerging therapeutic strategies
that target inflammatory cells and stroma within
the tumor microenvironment.
References and Notes
1. H. A. Burris 3rd et al., J. Clin. Oncol. 15, 2403 (1997).
2. C. E. Clark et al., Cancer Res. 67, 9518 (2007).
3. S. P. Schoenberger, R. E. Toes, E. I. van der Voort,
R. Offringa, C. J. Melief, Nature 393, 480 (1998).
4. S. R. Bennett et al., Nature 393, 478 (1998).
5. J. P. Ridge, F. Di Rosa, P. Matzinger, Nature 393,
6. R. R. French, H. T. Chan, A. L. Tutt, M. J. Glennie, Nat.
Med. 5, 548 (1999).
7. L. Diehl et al., Nat. Med. 5, 774 (1999).
8. E. M. Sotomayor et al., Nat. Med. 5, 780 (1999).
9. R. H. Vonderheide et al., J. Clin. Oncol. 25, 876
10. Materials and methods are available as supporting
material on Science Online.
11. A. K. Nowak, B. W. Robinson, R. A. Lake, Cancer Res. 63,
12. R. A. Lake, B. W. Robinson, Nat. Rev. Cancer 5, 397 (2005).
13. D. I. Gabrilovich, Lancet Oncol. 8, 2 (2007).
14. L. Zitvogel et al., J. Clin. Invest. 118, 1991 (2008).
15. S. R. Hingorani et al., Cancer Cell 7, 469 (2005).
16. K. P. Olive et al., Science 324, 1457 (2009).
17. T. Van Dyke, Nat. Med. 16, 976 (2010).
18. R. H. Vonderheide, Clin. Cancer Res. 13, 1083 (2007).
19. C. van Kooten, J. Banchereau, J. Leukoc. Biol. 67, 2 (2000).
20. N. Van Rooijen, A. Sanders, J. Immunol. Methods 174,
21. L. M. Coussens, Z. Werb, Nature 420, 860 (2002).
22. A. Mantovani, A. Sica, Curr. Opin. Immunol. 22, 231
23. E. M. Jaffee et al., J. Clin. Oncol. 19, 145 (2001).
24. We thank C. Abrams, E. Furth, C. June, B. Keith,
G. Koretzky, C. Simon, and B. Stanger for helpful
discussions. This research was supported by the Abramson
Family Cancer Research Institute of the University of
Pennsylvania School of Medicine (R.H.V.) and by NIH
grants P30 CA016520 (P.J.O. and R.H.V.) and K12
CA076931 (G.L.B.). The clinical study and part of the
preclinical studies were supported by funding from
Pfizer Corp (to R.H.V. and P.J.D). W. Song, D. Li, and
L. L. Sharp are employees of Pfizer Corp. P. J. O’Dwyer
discloses receiving honoraria from Pfizer; D. A. Torigian
owns stock in Pfizer. CP-870,893 is owned and
patented by Pfizer Corp., which manages its distribution.
Although now at a new address, R. D. Huhn was affiliated
with Pfizer Corp. during the conduct and analysis of
this study. Some of the clinical data were presented
at the 2010 American Society of Clinical Oncology Annual
Supporting Online Material
Materials and Methods
Figs. S1 to S15
29 September 2010; accepted 14 February 2011
Cortical Constriction During
Abscission Involves Helices
of ESCRT-III–Dependent Filaments
Julien Guizetti,1,2Lothar Schermelleh,3Jana Mäntler,4Sandra Maar,1Ina Poser,4
Heinrich Leonhardt,3Thomas Müller-Reichert,5,2* Daniel W. Gerlich1,2*
After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain
connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined
intermediate stages of abscission in human cells by using live imaging, three-dimensional
structured illumination microscopy, and electron tomography. We identified helices of
17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a
single stalk. The endosomal sorting complex required for transport (ESCRT)–III co-localized
with constriction zones and was required for assembly of 17-nanometer-diameter filaments.
Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at
the abscission site. The identification of contractile filament helices at the intercellular bridge
has broad implications for the understanding of cell division and of ESCRT-III–mediated fission of
large membrane structures.
bscission represents the very final step
of cell division in animal cells whereby
the two daughter cells are physically
severed from one another. The mechanism of
abscission is poorly understood (1–3), but it
plasma membrane wound healing (5). An al-
ternative model proposes that Golgi- (6) or re-
cycling endosome (7)–derived vesicles establish
membrane separation from within the intercel-
To clarify which events lead to abscission,
we imaged live HeLa cells stably expressing
enhanced green fluorescent protein (EGFP)–a-
tubulin (Fig. 1, A and B) (8). At the intercellular
bridge, microtubule bundles gradually narrowed
to a diameter of 0.97 T 0.13 mm (mean T SD; n =
17 cells) and then disassembled on one side
1Institute of Biochemistry, Department of Biology, Swiss
18, CH-8093 Zurich, Switzerland.2Marine Biological Labora-
tory (MBL), Woods Hole, MA 02543, USA.3Department of
Biology, Center for Integrated Protein Science Munich, Ludwig
Planegg-Martinsried, Germany.4Max Planck Institute for Mole-
Dresden, Germany.5Medical Theoretical Center, Medical Faculty
74, D-01307 Dresden, Germany.
*To whom correspondence should be addressed. E-mail:
email@example.com (D.W.G.); mueller-reichert@
25 MARCH 2011 VOL 331
on April 28, 2011