(Cd11b/CD18) Enhances Tumor Response to Radiation by Reducing Myeloid Cell Recruitment

Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment

G-One Ahna, Diane Tsenga,b, Cho-Hwa Liaoa, Mary Jo Doriea, Agnieszka Czechowiczb, and J. Martin Browna,1

aDivision of Radiation and Cancer Biology, Department of Radiation Oncology, and bInstitute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305

Edited by Napoleone Ferrara, Genentech, Inc., South San Francisco, CA, and approved March 26, 2010 (received for review October 2, 2009) Despite recent advances in radiotherapy, loco-regional failures are tions of myeloid cells, including adhesion, migration, chemotaxis, still the leading cause of death in many cancer patients. We have phagocytosis, and respiratory burst activity (7). Studies have previously reported that bone marrow-derived CD11b+ myeloid cells reported that antibodies to CD11b or CD18 inhibit these functions are recruited to tumors grown in irradiated tissues, thereby restoring (8) and in vivo administration of the antibodies reduce leukocyte the vasculature and tumor growth. In this study, we examined recruitment into various sites of inflammation (9). Given the whether neutralizing CD11b monoclonal antibodies could inhibit promising preclinical activities of CD11b/CD18 antibodies in the recruitment of myeloid cells into irradiated tumors and inhibit inhibiting myeloid cell adhesion onto endothelium, humanized their regrowth. We observed a significant enhancement of antitu- antibodies [LeukArrest; 23F2G (10)] have been developed and mor response to radiation in squamous cell carcinoma xenografts tested in patients with stroke, multiple sclerosis, or myocardial in mice when CD11b antibodies are administered systemically. His- infarction (11). However, although the antibodies showed excellent tological examination of tumors revealed that CD11b antibodies re- safety profiles, they lacked therapeutic efficacy (10, 11). duced infiltration of myeloid cells expressing S100A8 and matrix In this study, we used CD11b-neutralizing monoclonal anti- metalloproteinase-9. CD11b antibodies further inhibited bone bodies as a means to inhibit the recruitment of myeloid cells to marrow-derived cell adhesion and transmigration to C166 endothe- irradiated tumors. With systemic administration of these CD11b lial cell monolayers and chemotactic stimuli, respectively, to levels antibodies following local tumor irradiation, we observed a signif- comparable to those from CD11b knockout or CD18 hypomorphic icant enhancement of tumor response to radiation accompanied mice. Given the clinical availability of humanized CD18 antibodies, by a reduced infiltration of myeloid cells expressing MMP-9 and we tested two murine tumor models in CD18 hypomorphic or CD11b S100A8 into the tumors. We also observed that CD18 hypo- knockout mice and found that tumors were more sensitive to irradi- morphism, which had lowered CD11b surface expression myeloid ation when grown in CD18 hypomorphic mice but not in CD11b cells, significantly associated with the sensitivity of tumors to ra- knockout mice. When CD18 hypomorphism was partially rescued diation. Together, these results suggest that clinically available by reconstitution with the wild-type bone marrow, the resistance humanized antibodies against CD11b/CD18 could be useful as an of the tumors to irradiation was restored. Our study thus supports adjuvant therapy to radiotherapy. MEDICAL SCIENCES the rationale of using clinically available Mac-1 (CD11b/CD18) antibodies as an adjuvant therapy to radiotherapy. Results Radiation Inhibits Local Angiogenesis but the Vasculature Is Restored S100A8 | vasculogenesis | radiosensitivity in Recurrent Tumors Accompanied by Infiltrating Myeloid Cells. To determine the effects of local irradiation on angiogenesis, we adiotherapy plays a crucial role in cancer treatment, espe- first examined the histology of FaDu human head and neck Rcially for inoperable tumors. Recent advances, including squamous cell carcinoma xenografts grown in immune-deficient image-guided and intensity-modulated radiotherapy, leading to mice that were either unirradiated (control, 0 Gy), harvested higher and more precise dose delivery, has achieved superior shortly after 20 Gy of irradiation (IR 20 Gy), or had recurred treatment outcomes (1). However, despite these advances, re- after irradiation with 20 Gy, which took ≈2 months following currence of the primary tumors still remains the leading cause of irradiation [recurrent (2 mo)] (Fig. 1A). Tumor volumes were death of patients treated with radiotherapy (2). This finding ≈25 and 50% of the unirradiated control for irradiated tumors highlights the fact that we need an improved understanding of and recurrent tumors, respectively (Fig. 1A). By staining for the reasons for treatment failure. endothelial cells and pericytes with CD31 and α-smooth muscle We have previously shown that irradiated tumors, or tumors actin (α-SMA) antibodies, respectively, we found that the irra- grown in previously irradiated tissues (thereby mimicking re- diated tumors had significantly fewer endothelial cells and per- current primary tumors), recruit large numbers of bone marrow- icytes compared with the control tumors (Fig. 1 A and B). + derived CD11b myeloid cells expressing matrix metal- However, when the tumors had recurred after irradiation, the loproteinase-9 (MMP-9) (3). We further demonstrated that these number of endothelial cells had returned to control levels, al- MMP-9-expressing myeloid cells restored tumor vasculature and though the pericyte coverage was partially restored (Fig. 1 A and allowed tumor growth in the irradiated tissues of MMP-9 knockout B). To determine the myeloid cell contribution to the changes in (KO) mice, suggesting that these cells could be an important target in radiotherapy (3). There is strong evidence to suggest that tumor- fi + in ltrating CD11b myeloid cells promote angiogenesis, and do Author contributions: G-O.A., D.T., C.-H.L., M.J.D., A.C., and J.M.B. designed research; G-O.A., so by expressing various proangiogenic and chemoattractant D.T., C.-H.L., M.J.D., and A.C. performed research; G-O.A., D.T., C.-H.L., M.J.D., and A.C. ana- molecules, including VEGF (4), Bv8 (5), and S100A8 (6). How- lyzed data; and G-O.A. and J.M.B. wrote the paper. + ever, despite their tumor-promoting roles, targeting CD11b The authors declare no conflict of interest. myeloid cells as a cancer therapy has proven difficult. This article is a PNAS Direct Submission. CD11b (Mac-1, αMβ2) is the α-subunit of the predominant β2 1To whom correspondence should be addressed. E-mail: [email protected]. (CD18) integrin expressed on monocytes/macrophages and gran- This article contains supporting information online at www.pnas.org/cgi/content/full/ ulocytes (7). This subunit has been shown to mediate many func- 0911378107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0911378107 PNAS | May 4, 2010 | vol. 107 | no. 18 | 8363–8368 Downloaded by guest on September 25, 2021 + Control (0 Gy) IR 20 Gy (d14) Recurrent (2 mo) ﬁltration of CD11b myeloid cells, we determined whether anti-

A IPAD Tumor volume bodies to CD11b could reduce the myeloid cell recruitment and *** ) 3

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mm sensitize the tumors to irradiation. We harvested CD11b mono-

( 200 α e clonal antibodies (IgG2b isotype) from M1/70 hybridoma and ﬁrst 13DC m 150 ul

o 100 determined the dose and timing of the antibodies for in vivo ad- vromuT 50 IPAD IPAD ministration. We similarly studied Gr-1 antibodies (IgG2b isotype) 0 lort tnerru yG02R harvested from RB6-8C5 hybridoma. We ﬁrst observed that Gr-1 no b11DC ﬁ + + c C

I antibodies ef ciently depleted granulocytes (CD11b Gr-1 cells) eR at 24 h after a single i.p. administration; CD11b antibodies did not affect the leukocyte composition (Fig. S1A and Table S1). CD11b B CD31 α-SMA CD11b CD45 antibodies exhibited a complete epitope blockage with 100 μgat 6 20 4 10 )%(ytisn ** ** *** *** *** *** *** ** 24 h (Fig. S1 C and D), which was partially reversed at 72 h after 4 * administration (Fig. S1E). Therefore, to maintain constant epitope

e 10 2 5 dae 2 blocking of myeloid cells, we treated the mice with CD11b anti- r A bodies at 100 μg per mouse every 2 days. Gr-1 antibodies were 0 0 0 0 l lortnoC lo lortnoC tne tnerruceR tnerruceR tnerruceR y yG02RI y yG02RI o G02R G rt rtnoC

r administered to a separate group of animals in a similar manner. 02RI no r uc C I e To monitor epitope blockage by CD11b antibodies or granulo- R cyte depletion by Gr-1 antibodies, we sampled peripheral blood C Control (0 Gy) IR 20 Gy (d14) D CD31 α-SMA from the treated animals once every four days for FACS analysis )%(

24 10 *** H D

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e with CD11b antibodies following either 12 or 20 Gy of a single dose oH 0 0 fi lo lort of irradiation given locally to the tumors, we observed a signi cant yG02RII yG02RI r tnoC A no enhancement of tumor response to radiation (Fig. 2 ). Macro- C scopically, the CD11b antibody-treated tumors shrank dramati- Fig. 1. Local irradiation inhibits angiogenesis. (A) Staining of FaDu tumors cally, leading to nearly nonpalpable tumors by the end of the study grown in immunodeficient mice that were not irradiated (control, 0 Gy), (Fig. 2B) (12 of 16 tumor cures in CD11b antibody treatment and irradiated with 20 Gy and harvested at day 14 postirradiation [IR 20 Gy (d14)], 7 of 19 in the control groups in three independent experiments, or recurrent after 20 Gy of irradiation [recurrent (2 mo)]. The tumors were P < 0.05 by two-tailed Student’s t test). In contrast, Gr-1 antibodies stained for CD31 (red) and α-SMA (green) (Upper), and for CD11b (green) exhibited little or no effect (Fig. 2A), indicating that CD11b+Gr- − (Lower). Nuclei were stained with DAPI and are shown in blue. (Scale bars, 1+ cells play a less important role than CD11b+Gr-1 cells in 100 μm.) The bar graph shows the mean tumor volume. (B) Quantification of fl α in uencing tumor regrowth after irradiation. Further analysis of CD31, -SMA, CD11b, and CD45 shown in A. Symbols and error bars in A and + + + − B are the mean ± SEM for n ≥ 5 animals per group. *, **, and *** denote P < CD11b Gr-1 and CD11b Gr-1 cells by FACS revealed that 0.05, < 0.01, and < 0.001 by one-way ANOVA, respectively. (C) Immunostain- the former population of cells had slightly higher expression of ing of matrigel implanted in mice as in A, stained with CD31 (red) and α-SMA mature myeloid lineage markers, including macrophage colony- (green) antibodies. Hoechst 33342, a diffusion dye injected immediately be- stimulating factor-receptor (MCSF-R), 7/4, and F4/80 (Fig. S1 F fore the matrigel harvest, is shown in blue. (Scale bar, 100 μm.) (D) Quantifi- and G). We further tested CD11b antibodies in immunocompe- cation of the matrigel section from C as in B. Symbols and error bars are the tent C3H/HeJ mice bearing SCCVII tumors and observed that ± < ’ mean SEM for n = 4 animals per group. ***, P 0.001 by Student s t test. tumor regrowth after irradiation was also inhibited (Fig. 2C). We further tested the possibility that CD11b antibodies might the tumor vasculature, we stained the tumor sections with CD11b sensitize normal tissues to irradiation to a similar extent as that for myeloid cells and CD45 for all leukocytes. We observed that the levels of myeloid cells were increased in the irradiated tumors, and this increase was maintained in the recurrent tumors (Fig. 1 A A 12 Gy 20 Gy emulovromutevitaleR and B). Leukocyte levels were transiently increased in the irradi- emulovromutevitaleR 1 1 Control ated tumors but in the recurrent tumors had decreased to levels Gr-1 Ab B CD11b Ab 0.1 approaching that in the control tumors (Fig. 1 ). To separate the 0.1 effects of irradiation on existing versus new vasculature derived 0.01 0 10 20 30 0410 20 30 050 60 from angiogenesis, we implanted matrigel plugs in mice and irra- Days post irradiation Days post irradiation diated them with 20 Gy. In unirradiated (control) matrigels, there + was extensive penetration of CD31 endothelial cells into the B Control CD11b Ab C emulovromutevitaleR + 3 Control α CD11b Ab plugs, some of which were associated with -SMA pericytes yG02 C fi 2 (Fig. 1 ). In ltrating blood vessels were functional, as demon- 1

0 strated by Hoechst 33342 positivity, injected immediately before 0246810 the matrigel harvest. However, there were signiﬁcantly fewer en- Days post irradiation dothelial cells in the irradiated matrigel plugs (Fig. 1 C and D), Fig. 2. CD11b monoclonal antibody treatment enhances tumor response to demonstrating that irradiation inhibits the development of de radiation. (A) Growth of irradiated FaDu tumors with 12 Gy (Left)or20Gy novo vasculature. Overall, the results indicate that although ra- (Right) in mice treated with isotype control antibodies (control), Gr-1 anti- diation produces a major depletion of the tumor vasculature bodies (Gr-1 Ab), or CD11b antibodies (CD11b Ab) at 100 μg per mouse from (presumably because of killing of endothelial cells in the tumor and the fourth day following irradiation for every 2 days. (B) Photographs of inhibition of local angiogenesis from surrounding normal tissues), mice bearing FaDu tumors that had been irradiated with 20 Gy and treated with isotype control antibodies (control, Left) or CD11b antibodies (CD11b tumors that regrow following irradiation have a restored vascula- + Ab, Right) for up to 2 months. Tumors had regrown in the control group ture accompanied by an increased accumulation of CD11b my- (black arrowheads), whereas they became not palpable in the CD11b Ab eloid cells. group (black arrowheads indicate where the tumor had been originally implanted). (C) Growth of irradiated SCCVII tumors in C3H/HeJ mice with 15 CD11b Antibody Treatment Enhances Tumor Response to Radiation. Gy, followed by the control or CD11b antibodies. Symbols and error bars in A Given that recurrent tumors after radiation have an increased in- and C are the mean ± SEM for n ≥ 7 per group.

8364 | www.pnas.org/cgi/doi/10.1073/pnas.0911378107 Ahn et al. Downloaded by guest on September 25, 2021 observed with the tumors. To do this, we irradiated a region of CD11b Antibodies Reduce Infiltration of Myeloid Cells Expressing normal skin on the back of nontumor-bearing mice with 20, 25, or S100A8 and MMP-9. To determine whether the enhanced tumor 30 Gy and treated the animals with either saline or CD11b anti- response to radiation by CD11b antibodies was associated with bodies on the same schedule as that used for the tumor-bearing reduced myeloid cell infiltration in the irradiated tumors, we ex- mice. We found that CD11b antibodies did not sensitize, but rather amined the histology of FaDu tumors in mice treated with CD11b protected the normal skin from the radiation-induced skin re- antibodies or isotype control antibodies at 7 and 14 days after ir- action (P < 0.01 for median scores between the control and CD11b radiation. We found reduced levels of both CD11b+ myeloid cells antibody treated groups for all radiation doses, by two-tailed and CD45+ leukocyte levels in the CD11b antibody-treated Mann-Whitney t test) (Fig. S2A). This finding suggests that the tumors on day 7 after 20 Gy of irradiation (Fig. 3 A and B). enhanced tumor response to radiation by CD11b antibodies is not However, there was no significant difference in CD31+ endothe- a result of sensitizing normal tissues to radiation but rather of di- lial cells between the two groups at day 7, although α-SMA+ rect effects caused by CD11b antibodies to the irradiated tumors. pericytes were lower in the irradiated tumors treated with CD11b CD11b or Gr-1 antibodies alone, on the other hand, showed antibodies (Fig. 3 A and B). At day 14, CD11b antibody-treated no effects on the growth of nonirradiated tumors (Fig. S2B). tumors showed significantly reduced levels of CD45+ leukocytes, Histology of the antibody-treated tumors showed similar area although CD11b+ myeloid cells, CD31+ endothelial cells, and densities for CD31+ endothelial cells and α-SMA+ pericytes in α-SMA+ pericytes were similar to the control antibody treated all groups (Fig. S2 E and F). However, we observed less CD45+ tumors (Fig. S3B). leukocyte infiltration in the CD11b antibody-treated tumors, To further identify the myeloid subsets affected by CD11b whereas the Gr-1 antibody-treated tumors showed significantly antibody treatment in irradiated tumors, we first stained un- increased levels of CD45+ cells compared with the control irradiated (No IR) or recurrent (IR 20 Gy) FaDu tumors with tumors (Fig. S2 E and F). various myeloid markers including S100A8 and F4/80 in con-

A Control (d7) CD11b Ab (d7) B CD11b CD45 ) % ( y t i s n e d a e r A r e a d e n s i t y ( % ) 4 *** 10 I P I I I P A D A P I I P I *** P A P A A D D D 2

t a a t 5 b 1 1 D C C D D 1 1 b a r - r r - r i t i i t i n a n n a n 0 0 Control CD11b Control CD11b Ab Ab I P A D A P I I P P I I I P A D A P I A D A A A α

D CD31 -SMA ) % ( y t i s n e d a e r A r e a d e n s i t y ( % ) A M S - S M A A M A 3 20 * S - S S - α α 2 1 3 1 1 3 D C C D D 3 1 10 D D C C 1 MEDICAL SCIENCES 0 0 Control CD11b Control CD11b Ab Ab

S100A8 ) % ( n o i t a z i l a c o l - o c o - l o c a l i z a t i o Cn ( % ) D S100A8 MMP-9 DAPI

b 1 1 D C D 1 1 b 100 t n e n t 50 I R o c r c N e P 0 No IR IR 20 Gy

) S100A8 ) 9 % ( n ( 10 P - 100 % ** ( o i o M M t n e n t M M y t y G 0 2 0 G y t i s i a z i l a c o l - o c o - l o c a l i z a

n e n 5 50 d a d c r c R e e I r P

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y *** t t a r a t t a r - i t n a n t i - r a t i s i b 1 1 D C D 1 1 1 b - n e n i 2.5 t n a n t D d a d C 8 A 0 0 1 S 1 0 0 0 0 A A 8 8 A 0 0 1 S 1 1 0 0 A 8 e r

A 0 Control CD11b S S Ab

Fig. 3. CD11b monoclonal antibodies inhibit tumor infiltrating myeloid cells expressing S100A8 and MMP-9. (A) Immunostaining of FaDu tumors from Fig. 2A (20 Gy) harvested at 7 days (d7) after irradiation, stained for CD11b+ cells using CD11b (control tumors) or anti-rat (CD11b Ab-treated tumors) antibodies (Upper). (Lower) CD31 (red) and α-SMA (green) staining. Nuclei are shown in blue with DAPI staining. (B) Quantification of immunostaining in A as area densities for CD11b, CD45, CD31, and α-SMA. (C) Quantification of S100A8 immunostaining (Upper) and colocalization with CD11b (Lower) in unirradiated (No IR) or recurrent (IR 20 Gy) tumors in Fig. 1A.(D) Immunostaining of S100A8 (red) and MMP-9 (green) in unirradiated or recurrent tumors as shown in C. Quantification of colocalization between S100A8+ cells and MMP-9 is shown in the bar graph. (E) Immunostaining of d 7 FaDu tumors as shown in A for S100A8 (red) and CD11b (green; for control tumors), or anti-rat (green; for CD11b Ab-treated tumors) antibodies. The bar graph shows quantification of S100A8 immunostaining. (Scale bars for A, D, and E: 100 μm.) Symbols and error bars in B to E are the mean ± SEM for n ≥ 3 mice per group. *, **, and *** denote P < 0.05, < 0.01, and < 0.001 by Student’s t test, respectively.

Ahn et al. PNAS | May 4, 2010 | vol. 107 | no. 18 | 8365 Downloaded by guest on September 25, 2021 junction with CD11b. We found a strong colocalization (85–99%) with CD11b antibodies essentially abolished the increased migra- between CD11b and S100A8 markers (Fig. 3C), whereas F4/80 tion of the cells toward all of the chemotactic stimuli, resulting in the was poorly colocalized with CD11b in the tumors (Fig. S3A). number of migrated cells similar to the control group with no che- S100A8 showed significantly elevated levels in irradiated com- motaxis (Fig. 4C). A similar lack of migration toward the chemo- pared to unirradiated tumors (Fig. 3C), although F4/80 levels did tactic stimuli was seen in the bone marrow-derived cells isolated not significantly change (Fig. S3A). We further observed that from CD11b KO mice or CD18 hypomorphic mice (Fig. 4C). S100A8+ myeloid cells strongly expressed MMP-9, which revealed ≈80% of colocalization (Fig. 3D). Irradiated tumors treated with CD18 Hypomorphism Influences Tumor Sensitivity to Radiation. Be- CD11b antibodies exhibited a significant reduction of S100A8+ cause humanized CD18 antibodies are clinically available and have myeloid cells at day 7 (Fig. 3E) and day 14 (Fig. S3B), consistent shown similar efficacy in inhibiting myeloid cell functions to CD11b with the above results with CD11b staining (Fig. 3B and Fig. S3B). antibodies (14), we tested CD18 hypomorphic mice and CD11b KO mice for tumor response to radiation by using Lewis lung carcino- CD11b Antibodies Inhibit Adhesion and Transmigration of Bone mas (LLC) or MC38 colon adenocarcinomas, which are syngeneic Marrow-Derived Cells. To determine how CD11b antibodies re- to these mouse strains. We observed that tumors were more sensi- duced the infiltration of myeloid cells in the irradiated tumors, we tive to radiation when grown in CD18 hypomorphic mice, but not in first examined whether irradiation increases the expression of in- CD11b KO mice compared with the WT mice (Fig. 5A). As the tercellular adhesion molecule-1 (ICAM-1), a ligand of CD11b latter result was unexpected in view of the above data with CD11b integrin receptor (12), on endothelial cells. We observed that sur- antibodies, we examined the histology of the tumors. We found that face expression of ICAM-1 (Fig. 4A), but not vascular cell adhesion therewereinfiltrating S100A8+ myeloid cells in the irradiated molecule (VCAM-1) (Fig. S3C) was increased in irradiated C166 tumors from CD11b KO mice, despite of the genetic absence of endothelial cells in a dose- and time-dependent manner. We then CD11b (Fig. 5B). The levels of S100A8+ myeloid cells were similar examined the effect of CD11b antibodies on adhesion of carboxy- between CD11b KO and WT mice (Fig. 5B). We then examined fl uorescein succinimidyl ester (CFSE)-labeled bone marrow- CD11b levels in CD18 hypomorphic mice by subjecting the CFSE- derived cells onto C166 endothelial cells expressing endogenous labeled bone marrows isolated from CD18 hypomorphic mice to fi levels of ICAM-1. We observed that CD11b antibodies signi cantly FACS analysis. CD18 hypomorphic mice exhibited a significantly reduced bone marrow-derived cell adhesion onto the endothelial lower surface expression of CD11b on their bone marrow-derived B cells compared with the isotype control antibodies (Fig. 4 ). Fur- cells compared with the WT mice (Fig. 5C). To determine whether thermore, this effect was comparable to that of bone marrow- we could modulate tumor radiosensitivity by rescuing CD18 hypo- derived cells isolated from CD11b KO mice or CD18 hypomorphic morphism, we transplanted the WT bone marrow into nonwhole- B mice (Fig. 4 ), the latter resembling moderate levels of human body-irradiated CD18 hypomorphic mice once a week for 4 weeks. fi leukocyte adhesion de ciency (13). We further investigated whether At 6 weeks we examined the bone marrow reconstitution by pe- CD11b antibodies influence transmigration efficiency of the bone fi ripheral blood analyses and found that CD18 hypomorphism was marrow-derived cells by using a modi ed Boyden chamber assay. partially rescued by the WT bone marrow (Fig. 5 D and E). We We observed that pretreatment of the bone marrow-derived cells further observed that the response of MC38 tumors to radiation in the CD18 hypomorphic mice was reversed to the WT level in the CD18 hypomorphic mice reconstituted with the WT bone marrow + 24 hr 48 hr (Fig. 5F). Histology of the tumors showed that infiltrating CD11b A 20 Gy 20 Gy + 10 Gy 10 Gy G H 5 Gy 5 Gy myeloid cells (Fig. 5 )andCD18 cells (Fig. 5 )weredramatically 2.5 Gy 2.5 Gy 0 Gy 0 Gy Isotype Isotype increased in the CD18 hypomorphic mice partially reconstituted control ICAM-1 control ICAM-1 with the WT bone marrow, which otherwise was very low in the

Control CD11b Ab CD18 hypomorphic mice. These results are consistent with the *** I I P A D A P I

B P *** A d l e i e l d D *** antibody neutralization study, suggesting that inhibition of Mac-1 e f / f h

E S F C F S E 1 t S F S s n l l e l l i s e g e s i C (by CD11b antibodies or by CD18 hypomorphism) enhances tumor c d n e 0.5 a h h

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n 0 A l o b A b 1 b A b D O cells expressing S100A8. o r t n t r p y h 8 1 D 1 8 h y p K b 1 1 D 1 1 b K E S F C F S E S o C o F 1 C D C C C Discussion

No treatment 10 % serum VEGF M-CSF In this study, we show that CD11b monoclonal antibodies re- *** C *** d *** *** ﬁ l 4 4 4 4 *** e e duced the radiation-induced in ltration of myeloid cells into i f i

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s *** 3 i l l l 3 3 *** 3 s e e c squamous cell carcinoma xenografts in mice (Fig. 3). Further- g d

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n 0 0 0 0 l l l l o o o o b A b 1 1 1 b A b b 1 1 b A b b A b 1 b A b fi O O O O o o o o r t n t r r t n t r n t r r t n t r p y h y p p y h 8 1 8 h y p p y h 8 1 8 h y p p y h 8 1 8 h y p A b 1 1 1 b A K b 1 1 D 1 1 b K K K b 1 1 D 1 1 b K K b 1 1 D 1 1 b K response to irradiation (Fig. 2), with no signi cant effect on b 1 b o C o o C o C o o C o 8 1 1 D 1 1 D C D D C D D C D D C D D C D D C D D C D D C D C C C C unirradiated tumors (Fig. S2). These results are consistent with our earlier hypothesis (3) that under normal conditions, tumors Fig. 4. CD11b antibodies inhibit adhesion and transmigration of bone support their growth by local angiogenesis (by proliferation and marrow-derived cells. (A) Irradiated C166 endothelial cells analyzed by FACS showed up-regulation of ICAM-1 expression in a dose- (0–20 Gy) and time- migration of endothelial cells from nearby blood vessels) with (24 h, Left;48h,Right) dependent manner. (B) Fluorescent images of ad- little or no requirement for vasculogenesis from the bone mar- hered CFSE-labeled bone marrow cells (green) onto C166 endothelial cell row-derived circulating cells. However, as we have shown earlier monolayers (nuclei of the endothelial cells are shown in blue by DAPI with transplanted murine tumors (3), when the tumor and im- staining) that were either pretreated with isotype control (control) or CD11b mediately surrounding normal tissue are irradiated (which se- antibodies (CD11b Ab; Upper), or isolated from CD11b KO mice or CD18 verely inhibits local angiogenesis, as we show in Fig. 1), tumors hypomorphic (CD18 hypo) mice (Lower). (Scale bar, 100 μm.) Quantification fi become highly dependent on the vasculogenesis pathway to re- of the number of CFSE-positive cells per eld is shown on the right. (C) CFSE- store the tumor vasculature following its depletion by irradiation, labeled bone marrow derived cells as shown in B were incubated in modified Boyden chambers containing the culture media supplemented with no thereby supporting tumor recurrence. Therefore, both these and chemokine (no treatment), 10% serum, VEGF, or M-CSF. Symbols and error our prior data (3) demonstrate the importance of vasculogenesis, bars in B and C are the mean ± SEM for triplicate determinations in three and in particular CD11b cells, for restoring the tumor vascular independent experiments. ***, P < 0.001 by one-way ANOVA. and allowing tumor regrowth following irradiation.

8366 | www.pnas.org/cgi/doi/10.1073/pnas.0911378107 Ahn et al. Downloaded by guest on September 25, 2021 ﬁ + LLC MC38 although we observed an ef cient depletion of Gr-1 cells, which emulovromutevitaleR

A emulovromutevitaleR 4 4 WT were consistent with these reports, Gr-1 antibodies did not inhibit 3 3 CD11b KO CD18 hypo the growth of FaDu tumors in mice with (Fig. 2A) or without ir- − 2 2 radiation (Fig. S2). This result suggests that CD11b+Gr- 1 cells, 1 1 rather than CD11b+Gr-1+ cells, may play an important role in 0 0 restoring the vasculature after irradiation. Recent studies have 0481216 0102030 − Days since irradiation Days since irradiation suggested that CD11b+Gr-1 cells are the major proangiogenic B S100A8 CD11b DAPI C myeloid cells promoting tumor vascularization (16) and pro- gression to metastases (17). WT CD18 hypo

) 4 4 TW 10 10 Studies have shown that tumors recruit myeloid cells by se- %

( 4 3 3 s 10 10 ei ESFC 8A001S

t creting cytokines, including VEGF and M-CSF to facilitate neo- isned 2 2 2 10 10 101 101 vascularization (18, 19). VEGF and M-CSF signal through VEGF a er 0 100 100 a

OKb11DC 0 1 2 3 0 1 2 3 receptor-1 and M-CSF receptor, respectively, which are expressed TW 10 10 10 10 10 10 10 10 O Kb CD11b PE CD11b PE on myeloid cells, hence modulating their migrating abilities (20, 11DC 21). Once recruited to tumors, myeloid cells may produce various proangiogenic cytokines, including stromal-derived factor-1, TGF-β, and VEGF, as shown by macrophages cocultured with DECD18 hypo *** conditioned media derived from tumor cells (22). The present

)% *** ** WT CD18 hypo + WT BM 100 eviltn 104 104 104 ( ﬁ

s ndings that CD11b antibodies inhibit transmigration of bone ll CTIF81DC CTIF81DC CTIF81DC

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e 50 + 2 2 2 creP marrow-derived cells toward chemotactic stimuli thus suggest

10 10 10 8 1D 101 101 101 C 0 a mechanism by which the antibodies could attenuate the myeloid TW opyh81DC 0 0 0 opyh81DC

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100 101 102 103 100 101 102 103 100 101 102 103 B cell recruitment to tumors, thereby inhibiting neovascularization TW CD11b PE CD11b PE CD11b PE by vasculogenesis. + CD11b monoclonal antibodies are known to modify many func- MC38 tions of myeloid cells. Here, we showed that the antibodies inhibited F emulo 3 adhesion and migration of the bone marrow-derived cells to the v

romutevitaleR 2 WT CD18 hypo endothelial monolayers and chemotactic stimuli, respectively (Fig. + WT BM 1 CD18 hypo 4). Myeloid cells adhere on the endothelium via an interaction between CD11b (on the myeloid cells) and ICAM-1 (on the endo- 0 01020 thelium) (12). In this study, we observed that radiation directly Days since irradiation up-regulated ICAM-1 expression on the endothelial cells (Fig. 4), *** consistent with a study by Hallahan et al., who showed that irradi- G WT CD18 hypo CD18 hypo + WT BM ***

)% 3 a I IPA ation increased ICAM-1 expression on endothelial cells and that er PAD ( s a 2 eiti D b11D 54 54D leukocyte adhesion occurs concurrently with ICAM-1 expression sn 1 DC ed C C (23). Hence, we speculate that the enhanced ICAM-1 expression on b 0 11 TW op op MEDICAL SCIENCES

MB ﬁ D

yh8 the irradiated endothelium may be the rst step in recruiting mye- yh81 C T W+ 1DC loid cells into the irradiated tumors. Consistent with this hypothesis, DC Handschel et al. have reported that ICAM-1 expression in the en- ** WT CD18 hypo CD18 hypo + WT BM 6 ﬁ ﬁ H )%(seitisned dothelium and myeloid cell in ltration were signi cantly increased aera81DC

IP IPA 4 in radiation-induced inflammation in oral mucosa of head and AD D 2 neck cancer patients treated with radiotherapy (24). 81DC 0 Our study also shows that CD18 hypomorphism, but not genetic o TW opyh81DC pyh81DC MBTW+ deficiency of CD11b, affects tumor sensitivity to radiation (Fig. 5). The enhanced radiosensitivity of tumors in CD18 hypomorphism was associated with lowered levels of CD11b surface expression on Fig. 5. CD18 hypomorphism influences tumor response to radiation. (A) the bone marrow cells and subsequently lowered infiltration of Growth of irradiated LLC (Left) or MC38 (Right) tumors with 15 Gy in the WT, + CD11b KO (CD11b KO), or CD18 hypomorphic (CD18 hypo) mice. (B) Immu- CD11b myeloid cells into irradiated tumors (Fig. 5). The apparent nostaining of irradiated LLC in WT or CD11b KO as in A for S100A8 (red) and paradox of the lack of radiosensitivity of tumors in CD11b KO mice + CD11b (green). DAPI shows nuclear staining in blue. Quantification of was explained by the infiltration of S100A8 myeloid cells to the S100A8 area densities is shown in the bar graph. (C) FACS plots showing irradiated tumors in these mice. S100A8 is a myeloid-specificin- CFSE-labeled bone marrow cells isolated from WT or CD18 hypo mice for tracellular calcium binding protein (25) and has been reported to CD11b surface expression. (D) FACS analyses of the peripheral blood participate in various inflammatory responses, including vascular obtained from WT, CD18 hypo, or CD18 hypomorphic mice reconstituted injury (26), by regulating myeloid cell chemotaxis and adhesion (27). with the WT bone marrow cells (CD18 hypo + WT). (E) Quantification of + Furthermore, it has been demonstrated that S100A8 increases propidium iodide-negative, live CD18 cells (as highlighted in the red boxes + in D) in the WT, CD18 hypo, or CD18 hypo + WT mice. (F) MC38 tumor CD11b expression thereby facilitating adhesion of CD11b myeloid growth after irradiation with 15 Gy in WT, CD18 hypo, or CD18 hypo + WT cells onto its ligand (28). Our data demonstrating that CD11b mice shown in D and E.(G and H) Immunostaining of MC38 tumors in F for antibodies, but not the genetic deficiency of CD11b, could inhibit CD11b (red, G), CD18 (red, H), and CD45 (green, G). Nuclei are shown in blue infiltration of S100A8+ myeloid cells, therefore indicate that with DAPI staining. (Scale bars in B, G, and H: 100 μm.) The symbols and error S100A8 is required for CD11b cell surface expression in myeloid bars represent the mean ± SEM for n ≥ 5 per group (for A, E, and F)orn ≥ 3 cells and this is important for contributing to tumor resistance < < per group (for B, G, and H). ** and *** denote for P 0.01 and 0.001, to radiation. respectively, determined by one-way ANOVA. Relevant to a possible use with radiotherapy, we found that CD11b antibodies did not sensitize normal skin to irradiation but There is currently no therapy available to selectively deplete rather protected it from radiation-induced damage (Fig. S2A). In CD11b+ myeloid cells in humans. A number of preclinical studies agreement with this, Epperly et al. reported reduced levels of have reported that Gr-1 antibodies deplete myeloid cells and that pulmonary damage in mice treated with CD11b antibodies or in this depletion leads to an inhibition of tumor growth (15). However, mice transplanted with the bone marrow cells from CD18 hypo-

Ahn et al. PNAS | May 4, 2010 | vol. 107 | no. 18 | 8367 Downloaded by guest on September 25, 2021 morphic mice, and these effects were accompanied by a reduction Pharmingen for CD31 (biotin anti-mouse; clone Mec13.3), CD45 (biotin or of macrophage migration to the irradiated lungs (29). FITC-conjugated; clone 30-F11), CD11b (biotin or PE-conjugated; clone M1/ In conclusion, we present evidence that inhibiting the in- 70), and Gr-1 (FITC-conjugated; clone RB6-8C5); Sigma for α-SMA antibodies filtration of myeloid cells into irradiated xenografts by CD11b (FITC-conjugated, clone 1A4); Serotec for F4/80 antibodies (488 conjugated, monoclonal antibodies enhances their response to irradiation. clone CI:A3-1) and 7/4 (647 conjugated, clone 7/4); e-Bioscience for MCSF-R Because of their excellent safety profiles demonstrated in clinical (PE-conjugated; clone AFS98); and from R&D Systems for S100A8 (goat anti- trials and even protection of irradiated normal tissues, we believe mouse). The sections were stained as described previously (3) and examined that CD11b antibodies are an attractive candidate for further and digital fluorescence microscopic images were taken using a Leica × × evaluation as an adjunct therapy to radiotherapy. DM6000B microscope (with HC PL FLUOTAR 20 and 40 objective lenses with HC PLAN 10×/25 eyepieces) and Retiga Exi Q imaging camera. Images Materials and Methods were developed with Image-Pro-6.2 software. For analysis, images at 20× Mice and Tumors. All animal procedures were approved by Stanford’s Ad- objective were taken from nonnecrotic and viable tumor regions away from ministrative Panel on Laboratory Animal Care. Strains of the mice used are: the edge of tumors, and analyzed at least four sections per tumor or nu/nu immunodeficient nude mice (Charles River), CD11b KO mice matrigel and three to five animals per group by using ImageJ software for (B6.129S4-Itgamtm1Myd/J; Jackson Laboratories), CD18 hypomorphic mutant area densities as described elsewhere (30) or Image-Pro-6.2 software mice (B6.129S7-Itgb2tm1Bay/J; Jackson Laboratories), C57BL/6J (Jackson Lab- for colocalization. oratories), and C3H/HeJ (Jackson Laboratories). Female mice at 6 to 8 weeks of age were used. The mice were maintained in a germ-free environment Statistical Analysis. Statistical comparisons of data sets were performed by and had access to food and water ad libitum. two-tailed t test (Student’s t test or Mann-Whitney t test) or one-way Cells were maintained in Waymouth medium containing 15% FCS for FaDu ANOVA with Tukey posttest using Prizm software (V4.00 GraphPad Inc.). The cells or in DMEM plus 10% FCS for C166 endothelial cells [American Tissue data were considered to be significantly different when P < 0.05. Culture Collection (ATCC)], Lewis lung carcinoma (ATCC), MC38, and SCCVII cells. ACKNOWLEDGMENTS. We thank Drs. Douglas Hanahan and Chris Chiu – × 6 × 5 FaDu cells (6 8 10 cells per mouse), LLC (5 10 cells per mouse), MC38 (University of California, San Francisco) and Dr. Irving L. Weissman (Stanford × 5 × 5 (5 10 cells per mouse), and SCCVII (5 10 cells per mouse) were in- University) for hybridomas and technical discussions. The MC38 cell line was oculated and measured as described previously (3) in nude (for FaDu), C57BL/ obtainedfromDr. SamuelStrober (Stanford University). We alsothank Dr. Judith 6J, CD11b KO, and CD18 hypomorphic mice (for LLC or MC38), or C3H/HeJ Shizuru (Stanford University) and Dr. Seung-Jae Lee (POSTECH, Korea) for (for SCCVII) mice. Tumors were irradiated as described (3) when their vol- insightful suggestions for the manuscript. This study was supported by National umes reached ∼200 mm3 for FaDu and ∼100 mm3 for LLC, MC38, or SCCVII Institutes of Health Grant CA128873 (to J.M.B.) and a Gary Slezak/American tumors using a Phillips x-ray unit. Brain Tumor Association translational grant (to G-O.A.). D.T. is supported by a Howard Hughes Medical Institute Research Training Fellowship. C.-H.L. is a recipient of a Postdoctoral Research Abroad Program by the National Science fi fi Immunostaining and Quanti cation. The matrigel sections were xed with 4% Council of the Republic of China. A.C. is supported by the Medical Scientist PFA in PBS for 30 min at room temperature before staining. Tumors were Training Program at Stanford University School of Medicine, as well as a grant harvested as described earlier (3). Antibodies were purchased from BD from The Paul and Daisy Soros Fellowships for New Americans.

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