CANCER RESEARCH | TUMOR BIOLOGYAND IMMUNOLOGY

Integrin b4–Targeted Cancer Immunotherapies InhibitTumor Growth and Decrease Metastasis A C

Shasha Ruan1,2, Ming Lin1,3, Yong Zhu4, Lawrence Lum5, Archana Thakur5, Runming Jin3, Wenlong Shao6,Yalei Zhang6, Yangyang Hu7, Shiang Huang3, ElaineM. Hurt8, Alfred E. Chang1, Max S.Wicha1, andQiao Li1

ABSTRACT◥

Integrin b4 (ITGB4) has been shown to play an important role inthe regulation of cancer stem cells (CSC). Immune targeting ofITGB4 represents a novel approach to target this cell population,with potential clinical benefit. We developed two immunologicstrategies to target ITGB4: ITGB4 protein–pulsed dendritic cells(ITGB4-DC) for vaccination and adoptive transfer of anti-CD3/anti-ITGB4 bispecific antibody (ITGB4 BiAb)–armed tumor-draining lymph node T cells. Two immunocompetent mousemodels were utilized to assess the efficacy of these immunotherapiesin targeting both CSCs and bulk tumor populations: 4T1mammarytumors and SCC7 head and neck squamous carcinoma cell line.Immunologic targeting of ITGB4 utilizing either ITGB4-DC orITGB4 BiAb-T cells significantly inhibited local tumor growth andmetastases in both the 4T1 and SCC7 tumor models. Furthermore,the efficacy of both of these ITGB4-targeted immunotherapies wassignificantly enhanced by the addition of anti–PD-L1. Both ITGB4-

targeted immunotherapies induced endogenous T-cell cytotoxicitydirected at CSCs as well as non-CSCs, which expressed ITGB4, andimmune plasma–mediated killing of CSCs. As a result, ITGB4-targeted immunotherapy reduced not only the number of ITGB4high

CSCs in residual 4T1 and SCC7 tumors but also their tumor-initiating capacity in secondarymouse implants. In addition, treatedmice demonstrated no apparent toxicity. The specificity of thesetreatments was demonstrated by the lack of effects observed usingITGB4 knockout 4T1 or ITGB4-negative CT26 colon carcinomacells. Because ITGB4 is expressed by CSCs across a variety of tumortypes, these results support immunologic targeting of ITGB4 as apromising therapeutic strategy.

Significance: Immune targeting of ITGB4 may represent anovel approach to improve the efficacy of current cancerimmunotherapies.

IntroductionThe development of cancer immunotherapy represents one of the

most significant advances in oncology. Despite these successes, thebenefits of immunotherapy are limited to a subset of patients andtumor types. Furthermore, the durability of these responses is oftenlimited. There is increasing evidence that therapeutic resistance andtumor relapse may be mediated by a subset of tumor cells that displaystem cell properties (1–3). These cancer stem cells (CSC) lack expres-sion of differentiation antigens and may display inherent resistant to a

variety of immunotherapeutic approaches (2, 4). The ability of CSCs toescape recognition and elimination by the immune system maycontribute to the limited clinical efficacy of current cancer immu-notherapies. The targeting of shared CSC antigens represents anapproach to overcome these limitations.

Integrins are heterodimeric transmembrane receptors that mediateinteraction of cells with extracellular matrix components (5). Integrinb4 (ITGB4), which heterodimerizes exclusively with the a6 chain,functions as a receptor for the basement membrane protein laminin.ITGB4 expression is increased in a variety of malignancies includingbreast cancer cells (6, 7). ITGB4 is involved in and can enhancemultiple signaling pathways, including ErbB2 (8, 9), PI3K (10, 11),FAK/AKT (12, 13), and c-Met (14, 15), to promote tumor pro-gression (16). Exosome proteomics revealed the exosomal ITGB4was associated with lung metastasis (17, 18). Furthermore, upre-gulation of ITGB4 is an adverse prognostic marker in pancreaticductal adenocarcinoma (19) and breast cancer (20). Importantly,ITGB4 induces expansion of prostate tumor progenitors (21) andidentifies CSC-enriched populations from breast cancer cells (22). Itplays an important role in the metastasis and treatment resistance ofthese cells (23–25). We therefore hypothesized that immunologi-cally targeting ITGB4 might improve the efficacy of immunecheckpoint blockade by targeting the CSC population as well asbulk tumor cells.

In multiple tumor types, CSCs may be enriched by virtue of theirincreased expression of aldehyde dehydrogenase (ALDH) activity asaccessed by theAldefluor assay (26, 27). Inmousemodels ofmelanomaand head and neck cancer, we previously demonstrated the efficacyof a dendritic cell (DC) vaccine generated by pulsing these cells witha lysate of ALDHhigh CSCs (28, 29). This effect was mediated bycytotoxic CD8 T cells as well as antibodies that specifically targeted theCSC population. Furthermore, the therapeutic efficacy of ALDHhigh

head and neck CSC-DC vaccine was significantly augmented by

1Rogel Cancer Center, University ofMichigan, AnnArbor, Michigan. 2Departmentof Clinical Oncology, Renmin Hospital of Wuhan University, Wuhan, Hubei,China. 3Union Hospital, Tongji Medical College, Huazhong University ofScience and Technology, Wuhan, Hubei, China. 4Guangzhou ImproveMedical Instruments Co., Ltd. Guangzhou, Guangdong, China. 5Division ofHematology/Oncology, Department of Medicine, University of Virginia CancerCenter, Charlottesville, Virginia. 6The First Affiliated Hospital of GuangzhouMedical University, Guangzhou Institute of Respiratory Health, Guangzhou,China. 7Department of Rheumatology and Immunology, Tongji Hospital, TongjiMedical College, Huazhong University of Science and Technology, Wuhan,Hubei, China. 8Medimmune LLC, Gaithersburg, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Max S. Wicha, University of Michigan, 1500 E. MedicalCenter Dr., Ann Arbor, MI 48109. Phone: 734-936-6000; Fax: 734-232-5913;E-mail: mwicha@umich.edu; and Qiao Li, Phone: 734-615-1977; Fax: 734-998-2440; E-mail: qiaoli@umich.edu

Cancer Res 2020;80:771–83

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anti–PD-L1 administration (30). This immunotherapeutic augmen-tation was apparent in tumor models of advanced disease as well asthose simulating the adjuvant setting (30). Although these studiesdemonstrated the feasibility of generating immune responses againsttheCSCs, the clinical application of this approach is limited by the needto obtain tumor tissue to isolate CSCs from patient. An alternateapproach of targeting CSC-shared antigens has the potential forproviding an “off the shelf” reagent that can be utilized in patientswhose tumors express the antigen. Because ITGB4 is expressed inCSCs across multiple tumor types (17, 18, 21, 22), it is well suited forsuch immunologic targeting.

T-cell engaging bispecific antibodies (BiAb), which bring T effectorcells in contact with tumor cells, represent another approach forimmunologic targeting (31–33). We previously generated an anti-CD3/anti-CD133 bispecific antibody and bound it to cytokine-induced killer (CIK) cells as effector cells (BiAb-CIK) to targetCD133high CSCs. CIK cells bound with anti-CD3/anti-CD133 bispe-cific antibodies effectively targeted CD133high CSCs both in vitro andin vivo (34).

In this study, we explored two approaches for immunologictargeting of ITGB4 utilizing breast and head and neck cancermodels: ITGB4-DC vaccination and anti-CD3/anti-ITGB4 bispecificantibody–armed T cells adoptive transfer. We also demonstrated thatimmunologic targeting of ITGB4 enhanced the efficacy of anti–PD-L1checkpoint blockade in these models.

Materials and MethodsMice

All animal studies were approved by the Institutional Animal Careand Use Committee of the University of Michigan (Protocol numberPRO00006536 & 8556). Six- to 8-week-old female Balb/c mice andC3H mice were purchased from The Jackson Laboratory and CharlesRiver Laboratories.

Murine tumor cells4T1 is amurinemammary carcinoma cell line, which is syngeneic to

Balb/c mice (35). SCC7, a murine head and neck squamous carcinomacell line syngeneic to C3H mice, was described in our previouspublications (28). The murine colon carcinoma CT26 cell line is alsosyngeneic to Balb/c mice (36). 4T1, SCC7, and CT26 cells were kindlyprovided by Dr. Michael S. Sabel, Dr. Jeffrey S. Moyer, and Dr. JamesMoon, respectively. Luciferase-labeled 4T1 (4T1-Luc) cells were kindlyprovided by Dr. Max S. Wicha at the University of Michigan 2 yearsago. 4T1, 4T1-Luc, and SCC7 cells were utilized in this article overapproximately 2 years. Aliquots of these cells were frozen at thebeginning of the experiments. The passage number of 4T1, 4T1-Luc,and SCC7 cells is approximately 35. CT26 cells were used over the last3 months, with a passage number of approximately 10. All cell lineswere routinely tested for Mycoplasma contamination using MycoP-robe Mycoplasma Detection Kit (R&D Systems, Inc.), and any con-taminated cell line was treated with Plasmocin treatment (InvivoGen),confirmed by negative detection of Mycoplasma before being usedagain. The most recent testing was 8 months ago. All cell lines weregrown in complete medium (CM) consisting of RPMI1640 andsupplements (30).

Knockout of ITGB4 gene in 4T1 cell line via CRISPR/Cas9We generated ITGB4 knockout 4T1 (4T1-ITGB4KO) cells by using

Integrin b4 CRISPR/Cas9 KO Plasmid (Santa Cruz Biotechnology).According to themanufacturer's instructions, approximately 2.0� 105

4T1 cells were seeded in a 6-well tissue culture plate in 3 mL ofantibiotic-free standard growth medium per well 24 hours prior totransfection. At the 40% to 80% confluence, 4T1 cells were cotrans-fected with Integrin b4 CRISPR/Cas9 KO Plasmid and HDR Plasmidfor 72 hours and then selected with media containing puromycin(6 mg/mL). Knockout of ITGB4 in 4T1 cells was confirmed byWesternblot and flow cytometry as described below.

Western blottingTotal protein of CT26 or 4T1 cells or 4T1-ITGB4KO cells was

exacted by RIPA (Pierce Biotechnology) and concentrations deter-mined by Bio-Rad Protein Assay kit (Bio-Rad Laboratories). Equalamounts of protein lysates (30mg)were resolved by 6%SDS–PAGE (80V:�0.5 hours and 110V:�1.5 hours) and electrotransferred (320mA,2 hours) at 4�C onto polyvinylidene difluoride membranes. Blots wereblocked with 5% nonfat dry milk in Tris-buffered saline with 0.2%Tween-20 (TBST) for 1 hour at room temperature and incubatedovernight with primary murine ITGB4 antibody (Santa Cruz Biotech-nology; dilution: 1:250) or b-actin antibody (Invitrogen; dilution:1:1,000) at 4�C. After washing 3 times with TBST, the blots wereincubated with peroxidase-conjugated secondary antibody (Invitro-gen; dilution: 1:250) for 1 hour at room temperature. Blots werewashed 3 times and reactivity accessed by chemiluminescence(Thermo Fisher Scientific) using ChemiDoc Imaging System (Bio-Rad Laboratories).

Flow cytometryExpression of surface proteins was evaluated by flow cytometry.

Anti-ITGB4 antibody (Invitrogen) was used to define the ITGB4expression. Other antibodies include anti–PD-L1 (BD Biosciences),anti-IgG, and isotype controls (both from BD Biosciences). Stainedcells werefixedwith 4%paraformaldehyde (AlfaAesar), detected usingthe BD LSRFortessa Flow Cytometer (BD Biosciences), and analyzedusing the FlowJo software (Treestar).

ALDEFLUOR assayThe ALDEFLUOR Kit (StemCell Technologies) was used to isolate

ALDHhigh CSCs/ALDHlow non-CSCs from the 4T1 and SCC7 cells asdescribed previously (28). We stained cells with 7-actinoaminomycin-D to exclude dead and late apoptotic cells to sort CSCs with highviability. We used the top 10% of ALDHhigh cells and the lowest 10%ALDHlow cells for the subsequent experiments.

Murine integrin beta 4 protein–pulsed DC vaccineMurine integrin beta 4 (mITGB4) proteins were synthesized by

LifeTein company. Murine bone marrow derived DCs (BMDC) wereexpanded with GM-CSF (GenScript) and 2-mercaptoethanol (Gibco)as previously described in our laboratory (30, 37). Note that 1 � 106

BMDCs were loaded with 20 mg mITGB4 protein and cocultured at37�C overnight to generate the mITGB4-pulsed DC vaccine(mITGB4-DC).

Murine anti-CD3/anti-ITGB4 BiAb–armed tumor-draininglymph node T cells

Murine anti-ITGB4 monoclonal antibody (Invitrogen) and anti-CD3 antibody (BD Biosciences) were coupled by Dr. Lawrence G.Lum Lab (University of Virginia) to produce murine anti-CD3/anti-ITGB4 BiAbs (mITGB4 BiAb). In order to induce 4T1 tumor-draining lymph node (TDLN), 1 � 106 4T1 or 4T1-Luc tumor cellsin 0.1 mL PBS were injected s.c. into the lower flanks of immuno-competent Balb/c mice. To induce SCC7 TDLN, 1 � 106 SCC7

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tumor cells in 0.1 mL PBS were injected (s.c.) into the lower flanksof immunocompetent C3H mice. We used the same method toinduce CT26 TDLN in Balb/c mice. Nine days after cells’ inocu-lation of the tumor cells, the TDLNs were harvested, and single-cellsuspensions were prepared mechanically (38). TDLN cells wereactivated with anti-CD3 mAb and anti-CD28 mAb immobilized6-well tissue culture plates for 2 days and expanded in CM contain-ing 200 ng/mL of human rIL2 (PeproTech Inc.) for 3 days togenerate activated/expended TDLN T cells. Note that 1 � 106

activated TDLN T cells were incubated with 50 ng of mITGB4BiAb for 1 hour at 4�C to generate murine ITGB4 BiAb–armedTDLN T cells (TDLN T-mITGB4 BiAb; ref. 39).

Tumor models and treatment protocolsMinimal tumor model treated with mITGB4-DC vaccine

4T1 cells were injected into the mammary fat pad of Balb/c mice.SCC7 cells were injected into the C3H mice subcutaneously. Thetumor-bearing animals were then divided into different groups (n ¼5). Twenty-four hours (1 day) after tumor injection, PBS,mITGB4-DCvaccine, anti–PD-L1 (MedImmune LLC), and the combination treat-ment of mITGB4-DC vaccine plus with anti–PD-L1 antibody wereadministered respectively. The vaccination was repeated on day 8.Anti–PD-L1 was administrated 3 times after each DC vaccine. Eachvaccine contained 2 � 106 DCs per mouse, administered s.c., andanti–PD-L1 was administered i.p. in minimal tumor models. Fortumor monitoring, the long and short diameters of tumor masseswere measured 3 times per week. The volumes were calculated as:tumor volume ¼ (width2 � length)/2.

Established tumormodel treatedwithmITGB4BiAb–armedTDLNT cells

Fourteen-day 4T1 or 4T1-Luc, or SCC7 tumor–bearing animalswere divided into different groups (n ¼ 5): PBS, TDLN T cells,ITGB4 BiAb–armed TDLN T cells, anti–PD-L1, ITGB4 BiAb–armed TDLN T cells plus with anti–PD-L1 isotype control, andITGB4 BiAb–armed TDLN T cells plus anti–PD-L1, respectively.The mITGB4 BiAb–armed 4T1 TDLN T cells or SCC7 TDLN Tcells were transferred on day 14 and day 21 into 4T1 and SCC7tumor–bearing hosts, respectively. Each mouse was injectedwith 1 � 106 mITGB4 BiAb–armed TDLN T cells via tail vain.Anti–PD-L1 was administered twice after each T-cell transfer.Tumor growth was monitored in a same way as the tumor treatedby the DC vaccine. In addition, luciferase imaging was performedusing the PerkinElmer's IVIS Spectrum imaging system.

Experimental lung metastasis model treated with mITGB4-DCvaccine or mITGB4 BiAb–armed TDLN T cells

An experimental lung metastasis model was utilized to investigatetumor metastasis (40–42). Balb/c mice were injected i.v. with 4T1-Luccells via tail vain, whereas C3Hmice were injected i.v. with SCC7 cells,to produce experimental lung metastases. Mice were then divided intotreatment and control groups. mITGB4-DC vaccine was administrat-ed on days 1 and 8, respectively, with each vaccine being followed byanti–PD-L1 injection. mITGB4 BiAb–armed TDLN T cells weretransferred on days 3 and 10 with each T-cell transfer being followedby anti–PD-L1 injection. At the end of the experiments, lungs wereharvested to enumerate metastases.

Purification and culture of host T cellsSpleens were harvested from animals subjected to various treat-

ments at the endof the experiments. SplenicCD3þTcells were selected

byCD3MicroBeads (Miltenyi Biotec), activated by anti-CD3 and anti-CD28 (both from BD Biosciences), and then expanded with IL2 togenerate cytotoxic T cells (CTL; ref. 28).

CTL or TDLN T-cell cytotoxicityCTL or TDLN T-cell–mediated cytotoxicity was tested using the

CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega)according to the manufacturer's protocol. CTLs or TDLN T cells werecocultured with target cells (e.g., unsorted/ALDHhigh/ALDHlow 4T1,SCC7 cells, 4T1-ITGB4KO, or CT26 cells) for 6 hours, and then thesupernatants were harvested to measure lactate dehydrogenase. Thefollowing formula was used to calculate cytotoxicity: % Cytotoxicity¼(Experimental–Effector spontaneous–Target spontaneous)/(Targetmaximum–Target spontaneous) � 100.

Binding of immune plasma to tumor cellsPlasma was collected from treated hosts at the end of the

experiments. IgG levels were quantified using ELISA. Unsorted/ALDHhigh/ALDHlow 4T1 or SCC7 cells were incubated with theplasma containing equal quantities of IgG for 60 minutes on ice.Cells were washed twice and incubated with anti-mouse IgG(eBioscience) for 30 minutes on ice. Cells were then washedtwice, and their binding to plasma IgG was detected by flowcytometry.

Complement-dependent cytotoxicityCell lysis mediated by antibodies in immune plasmawas assessed by

incubation of 105 viable ALDHhigh/ALDHlow 4T1 or SCC7 cells withequal quantities of IgG on ice for 1 hour, followed by culturing in thepresence of rabbit complement (Calbiochem) in a 37�C water bath foranother 1 hour. Viable cells were then counted after trypan blue(Gibco) staining to calculate cell lysis: % of viable cells ¼ viable cellsafter plasma/complement incubation/105. Lower percentage of viablecells indicates more cell lysis.

Tumorigenicity of residual ALDHhigh CSCs or whole residualtumor cells

Treated primary tumors were harvested at the end of experi-ments and processed into single-cell suspensions, using 1 �collagenase/hyaluronidase solution (StemCell Technologies).Residual ALDHhigh CSCs were isolated from the cell suspension.We ensured that viable ALDHhigh CSCs or whole residual tumorcells were injected by staining these cells with Trypan Blue beforecell counts and injection. Tumor growth was then monitored (28).Similarly, whole residual tumor cells were injected into thesyngeneic mice to compare the tumorigenicity of the residual cellsafter different treatments.

Statistical analysisAll data are presented as mean� SEM. Differences between groups

were analyzed by one-way ANOVA or two-way ANOVA. The soft-ware GraphPad Prism 7 & 8 (GraphPad Software Inc.) was used toperform the statistical data analysis. Significant differences among datagroups were assigned when P< 0.05.

ResultsExpression of ITGB4 and PD-L1 on 4T1 and SCC7 cells

We analyzed ITGB4 expression in 4T1 murine breast cancer andSCC7 murine squamous cancer cell lines by flow cytometry. Wecompared the expression of ITGB4 on bulk cells as well as CSCs,

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which have been previously defined at ALDHhigh cells (30, 37). Wefound that 4T1 cells highly expressed ITGB4 in bulk unsorted cells(92.1%), in isolated ALDHhigh cells (84.2%), as well as inALDHlow cells(83.7%; Fig. 1A and B). In contrast in SCC7 cells, the expression ofITGB4 on ALDHhigh cells (37.1%) was significantly (P < 0.05) greaterthan that onALDHlow cells (26.5%; Fig. 1C andD). These results allowus to determine the effects of ITGB4 targeting on both bulk and CSCpopulations.

We also accessed the expression of the immune checkpointmolecule PD-L1 on these cell populations. The percentage ofPD-L1–expressing unsorted, ALDHhigh and ALDHlow 4T1 cellswas 21.8 (16.8 þ 5.0)%, 23.6 (21.5 þ2.1)%, and 25 (16.4 þ8.6)%respectively (Fig. 1E). For SCC7 cells, 44.6 (42.3 þ2.3)% ofunsorted cells, 22.4 (22.0 þ 0.4)% of ALDHhigh cells, and 18.4

(17.6 þ 0.8)% of ALDHlow cells expressed PD-L1 (Fig. 1F). Thesedata provide the rationale to determine whether the addition ofanti–PD-L1 checkpoint blockade can augment the effects of ITGB4targeting.

mITGB4-DC vaccination inhibited both local tumor growth andlung metastases in both 4T1 and SCC7 models

We have previously shown that ALDHhigh CSC lysate-pulsed DCvaccine conferred significant antitumor immunity in both tumorprotection (28) and therapeutic models (29). To determine whetherITGB4 presented by DC can effectively target ITGB4-expressingtumors, we prepared an mITGB4-DC vaccine and treated mice withminimal disease burden in 4T1 or SCC7 tumor–bearing mice. Inthese therapeutic experiments, some groups were also treated with

Figure 1.

Expression of ITGB4 and PD-L1 on 4T1 and SCC7 unsorted cells, ALDHhigh cells, and ALDHlow cells. A, 4T1 cells highly expressed ITGB4 in unsorted, ALDHhigh, andALDHlow cell populations. B, Statistical data showed no difference in the expression of ITGB4 in ALDHhigh and ALDHlow 4T1 subpopulations (P > 0.05). C, SCC7 cellsalso expressed high level of ITGB4 on unsorted cells, and its expression was significantly higher on ALDHhigh CSCs than on ALDHlow non-CSCs. D, Statistically, ITGB4cell-surface abundance onALDHhigh SCC7 cellswas approximately 1.5-fold higher than that onALDHlow SCC7 cells (P<0.05).E andF,PD-L1 expression on the 4T1 (E)and SCC7 (F) cells. Flow cytometry was performed at least twice, and one presensitive datum is shown for each experiment.

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Figure 2.

mITGB4-DC vaccine in ITGB4-targeted immunotherapy.A, Protocol of the 4T1 and SCC7 therapeuticminimal tumormodels. Treatment groupswere as follows: PBS,mITGB4-DC vaccine, anti–PD-L1, and the combination of mITGB4-DC vaccine and anti–PD-L1 (n ¼ 5). In 4T1 model, the administration of mITGB4-DC vaccineinhibited both local tumor growth (B andC) and spontaneous lungmetastases (D and E); coadministration of anti–PD-L1 mAb significantly enhanced the therapeuticeffectiveness (B–E). Pictures of local tumors (C) and spontaneous lung metastases (E) from treated animals bearing 4T1 tumors are shown. F and G, mITGB4-DCvaccine and/or anti–PD-L1 inhibited the local tumor growth in SCC7 therapeutic minimal tumor. H, SCC7 experimental lung metastasis model using mITGB4-DCvaccine. I and J,mITGB4-DC vaccine and/or anti–PD-L1 significantly inhibited lungmetastases in SCC7 experimental lungmetastasismodel. The experiment of 4T1 orSCC7 therapeutic minimal models was repeated four times.

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anti–PD-L1 mAb (Fig. 2A). In the 4T1 model, mITGB4-DC vacci-nation significantly inhibited local tumor growth (Fig. 2B and C) aswell as the development of spontaneous lung metastases (Fig. 2Dand E). In addition, the therapeutic effectiveness was significantly(P < 0.05) enhanced by anti–PD-L1 coadministration (Fig. 2B–E). Inthe SCC7 model, mITGB4-DC vaccination alone or in combinationwith anti–PD-L1 inhibited local tumor growth (Fig. 2F and G).

Unlike 4T1, SCC7 does not spontaneously metastasize whenimplanted subcutaneously. In order to determine the effect ofmITGB4-DC vaccine on tumor metastases in this model, we exper-imentally induced lungmetastases by i.v. injection (41–43). SCC7 cellswere injected via tail vein into syngeneic C3H mice, followed bymITGB4-DC vaccination with or without anti–PD-L1 administration(Fig. 2H). As shown in Fig. 2I and J, the mITGB4-DC vaccinesignificantly inhibited lung metastases, and this inhibition was signif-icantly enhanced by anti–PD-L1 administration.

mITGB4 BiAb–armed TDLN T-cell transfer inhibited local tumorgrowth and lung metastases

To confirm that ITGB4 can serve as an effective target forcancer immunotherapy, we evaluated a different immunologicstrategy to target ITGB4. To this end, we generated murine anti-CD3/anti-ITGB4 bispecific antibodies (mITGB4 BiAb) andtested the direct cytotoxicity of activated mITGB4 BiAb–armedTDLN T cells on ALDHhigh and ALDHlow 4T1 or SCC7 cellsin vitro. mITGB4 BiAb–armed TDLN T cells killed ALDHhigh

and ALDHlow 4T1 cells similarly, higher than nonarmed TDLNT cells, and the killing could be enhanced by the addition ofanti–PD-L1 in culture (Fig. 3A). However, mITGB4 BiAb–armedTDLN T cells killed more ALDHhigh SCC7 cells than ALDHlow

SCC7 cells, and the killing was significantly enhanced by anti–PD-L1 as well (Fig. 3B).

Based on these in vitro data, we proceeded to test mITGB4 BiAb–armed TDLN T cell transfer in mouse models bearing established(14 day) tumors (Fig. 3C). Local tumor growth was significantlyreduced after mITGB4 BiAb–armed TDLN T-cell transfer in the4T1 (Fig. 3D and E) and the SCC7 (Fig. 3F and G) models.Combining anti–PD-L1 therapy resulted in significant reduction inlocal tumor burden compared with mITGB4 BiAb-T cells’ transferalone (Fig. 3D–G).

We then tested experimental lung metastases of 4T1 and SCC7tumors treated with mITGB4 BiAb–armed TDLN T cells. 4T1-Luccells and SCC7 cells were injected via tail vein into syngeneic Balb/cmice and C3H mice, respectively, followed by treatments withmITGB4 BiAb-TDLN T cells and/or anti–PD-L1 (Fig. 3H). Lungmetastases were significantly reduced by mITGB4 BiAb-TDLN T-celltransfer, and this therapeutic effectiveness was significantly augment-ed by combiningwith anti–PD-L1mAb in both 4T1 (Fig. 3I and J) andSCC7 (Fig. 3K and L) models.

Both mITGB4-DC vaccination and mITGB4 BiAb–armed TDLNT-cell transfer specifically targeted ITGB4

To determine the specificity of ITGB4 targeting, we generatedITGB4 knockout 4T1 (4T1-ITGB4KO) cells and in addition assessedtreatment effects in CT26 murine colon carcinoma cells, which do notexpress ITGB4. Lack of ITGB4 expression in these cells was confirmedby Western blot (Fig. 4A) and flow cytometry (Fig. 4B). We treated4T1, 4T1-ITGB4KO, or CT26 tumor–bearing mice with mITGB4-DCvaccine in minimal tumor model (similar to Fig. 2A) or with TDLNT-mITGB4 BiAb in established tumor model (similar to Fig. 3C).Compared with PBS group, mITGB4-DC vaccine (Fig. 4C) or TDLN

T-mITGB4 BiAb (Fig. 4D) significantly (P < 0.05) inhibited the tumorgrowth of 4T1 cells. However, mITGB4-DC vaccine (Fig. 4C) orTDLN T-mITGB4 BiAb (Fig. 4D) did not affect growth of4T1-ITGB4KO cells or CT26 cells. Interestingly, knockout of ITGB4significantly (P < 0.05) reduced the tumor growth of 4T1 cells evenwithout treatment (Fig. 4C andD). This suggests that ITGB4 plays animportant biological role in these cells.

To confirm the specific targeting of ITGB4 by mITGB4-DC vaccineor TDLN T-mITGB4 BiAb, we performed in vitro killing experimentsusing splenic CD3þ T cells from animals subjected to vaccination orthe mITGB4 BiAb–armed TDLN T cells. CD3þ T cells were isolatedfrom the spleens of Balb/c mice treated with PBS or mITGB4-DCvaccine, respectively. After activation and expansion as describedin Materials and Methods, these T cells were cocultured with 4T1,4T1-ITGB4KO, or CT26 cells for 6 hours respectively. Splenic CD3þ

T-cell–mediated cytotoxicity was measured using the CytoTox 96Non-Radioactive Cytotoxicity Assay kit (Promega). We found(Fig. 4E) that although splenic CD3þ T cell from PBS-treated micekilled 4T1, 4T1-ITGB4KO, and CT26 cells equally, splenic CD3þ T cellfrom mITGB4-DC vaccine–treated mice killed 4T1 cells significantly(P < 0.05) more than splenic CD3þ T cell from PBS-treated mice, andsplenic CD3þ T cell from mITGB4-DC vaccine–treated mice killed4T1 cells significantly (P < 0.05) more than 4T1-ITGB4KO and CT26cells. These data thus confirm thatmITGB4-DCvaccine inducedT-celltargeting of ITGB4, and this targeting is ITGB4 specific (Fig. 4E).

In separate experiments, nonarmed TDLN T cells and mITGB4BiAb–armed TDLN T cells were cocultured with 4T1, 4T1-ITGB4KO,or CT26 cells, and killing assays were performed by harvesting thecocultured supernatants to measure lactate dehydrogenase. We found(Fig. 4F) that although nonarmed TDLN T cells killed 4T1,4T1-ITGB4KO, and CT26 cells equally, armed TDLN T-mITGB4 BiAbkilled 4T1 significantly (P < 0.05) more than nonarmed TDLN T cells,and TDLNT-mITGB4 BiAb cells killed 4T1 cells significantly (P < 0.05)more than their killing of 4T1-ITGB4KO and CT26 cells. These datathus further confirm that TDLN T-mITGB4 BiAb cells target ITGB4-expressing tumor cells, and this targeting is ITGB4 specific (Fig. 4F).

Together, our data demonstrated that both mITGB4-DC vaccineand mITGB4 BiAb–armed T-cell adoptive transfer specifically targetITGB4-expressing tumor cells in vitro and in vivo.

ITGB4-targeted immunotherapies conferred host immunityagainst ALDHhigh CSCs and ALDHlow non-CSCs

To understand the mechanism(s) underlying therapeutic efficacy,we harvested host spleens and immune plasma at the end of thetherapeutic experiments from 4T1 and SCC7models, respectively, andevaluated host T- and B-cell responses.

Splenic CD3þ T cells isolated from the 4T1-bearing mice(from Fig. 2B) treated with mITGB4-DC vaccineþ anti–PD-L1 mAbkilled unsorted, ALDHhigh, and ALDHlow 4T1 cells in vitro, signifi-cantly (P< 0.05) higher thanCD3þT cells isolated frommice subjectedto each monotherapy or PBS control (Fig. 5A). Of note, similar killingwas observed in ALDHhigh 4T1 cells and ALDHlow 4T1 cells. Inaddition, splenic CD3þ T cells isolated from the 4T1-bearing mice(from Fig. 3D) treated with mITGB4 BiAb–armed TDLN T cells þanti–PD-L1 mAb killed unsorted, ALDHhigh, and ALDHlow 4T1 cellsin vitro, significantly (P < 0.05) higher than CD3þT cells isolated fromthe mice subjected to each monotherapy and PBS control (Fig. 5B).Again, such killing was comparable against ALDHhigh 4T1 cells versusALDHlow 4T1 cells.

We also accessed the induction of humoral anti-CSC immunityby ITGB4-DC–based immunotherapy. To this end, we collected

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Figure 3.

ITGB4-targeted immunotherapy using mITGB4 BiAb–armed TDLN T cells via adoptive transfer. A and B, In vitro CTL activity of TDLN T cells cocultured with 4T1 (A)and SCC7 (B) ALDHhigh versus ALDHlow populations. C, Experimental protocol of the therapeutic 4T1- and SCC7-established (day 14) tumor models by adoptivetransfer ofmITGB4BiAb–armedT cells. Treatment groups included: PBS, TDLNT cells, TDLNT-mITGB4BiAb, anti–PD-L1, TDLNT-mITGB4BiAbþ anti–PD-L1 isotype,and TDLN T-mITGB4 BiAbþ anti–PD-L1 (n¼ 5).D, TDLN T-mITGB4BiAb significantly inhibited tumor growth in therapeutic 4T1model, whichwas enhanced by anti–PD-L1. E, Luminescence imaged by IVIS at the end of experiments to display tumor size in treated mice of the therapeutic 4T1 model. F and G, In therapeutic SCC7model, TDLN T-mITGB4 BiAb inhibited tumor growth, and coinjection and anti–PD-L1 significantly boosted the therapeutic effectiveness. H, Experimental lungmetastasis protocol. I–L, In both 4T1 and SCC7 experimental lungmetastasismodels, TDLN T-mITGB4BiAb significantly suppressed themetastases. Statistics (I) andrepresentative photos (J) of 4T1 tumors from treated Balb/c mice are shown. Statistics (K) and representative photos (L) of SCC7 tumors from treated C3Hmice areshown. The therapeutic experiments on 4T1 and SCC7 local tumors were repeated three times.

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immune plasma from the peripheral blood of 4T1 (from Fig. 2B) orSCC7 (from Fig. 2F) bearing mice subjected to treatments. We firstaccessed the specificity of ITGB4-DC vaccine–primed antibody bybinding assays of the plasma to tumor cells. Immune plasmafrom Balb/c mice that received the monotreatment of ITGB4-DCvaccine or anti–PD-L1 bound to 4T1 cells (35.1% and 34.1%respectively) more effectively than the immune plasma collectedfrom PBS-treated (26.7%; Fig. 5C). The binding activity wasenhanced by the ITGB4-DC vaccine þ anti–PD-L1 mAb treatment(38.5%; Fig. 5C). In the SCC7 model, immune plasma harvestedfrom ITGB4-DC vaccinated mice bound to unsorted, ALDHhigh,

and ALDHlow SCC7 cells more than the immune plasma harvestedfrom anti–PD-L1-treated mice or from PBS-treated controls,respectively, and the combination treatment did not show additivebinding effect (Fig. 5D). Notably, equal amounts of IgG in theimmune plasma collected from the ITGB4-DC vaccine þ anti–PD-L1-treated C3H mice bound to unsorted SCC7 (48.9%) more thanits binding to ALDHhigh and ALDHlow SCC7. However, the bindingto ALDHhigh SCC7 CSCs (28.5%) was higher than that to ALDHlow

SCC7 non-CSCs (18.8%; Fig. 5D).To examine the consequence of the binding of immune plasma to

tumor cells, we utilized antibody and complement-dependent

Figure 4.

BothmITGB4-DC vaccination andmITGB4 BiAb–armed TDLN T-cell transfer specifically target ITGB4.A andB,Confirmation of ITGB4 knockout in 4T1-ITGB4KO cellsand lack of expression of ITGB4 inCT26 cells viaWestern blot (A) and flowcytometry (B).C andD, ITGB4-targeted cancer immunotherapies specifically target ITGB4in vivo. mITGB4-DC (C) and TDLN T-mITGB4 BiAb (D) significantly inhibited the tumor growth of 4T1 cells, but not 4T1-ITGB4KO or ITGB4-negative CT26 tumors.Experimental groups included 4T1, 4T1-ITGB4KO, or CT26 tumor–bearing mice treated with PBS versus mITGB4-DC vaccine in minimal tumor model (C) or treatedwith PBS versus TDLNT-mITGB4BiAb in established tumormodel (D).E,CD3þT cells isolated from the spleensof Balb/cmice treatedwithmITGB4-DC vaccine killedITGB4-expressing 4T1 cells specifically in vitro. F, TDLN T-mITGB4 BiAbmediated significant greater cytotoxicity to ITGB4-expressing 4T1 cells than to 4T1-ITGB4KO

cells or ITGB4-negative CT26 cells in vitro.

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cytotoxicity (CDC) assays in both 4T1 and SCC7 models. ITGB4-DCvaccine–primed immune plasma killed both ALDHhigh and ALDHlow

4T1 cells significantly, and the ITGB4-DC vaccine plus anti–PD-L1-primed immune plasma killed both ALDHhigh and ALDHlow 4T1 cellssignificantly higher than the immune plasma collected from all theother groups (Fig. 5E). In the SCC7 model, ITGB4-DC vaccine–primed immune plasma killed ALDHhigh SCC7 cells more thanALDHlow SCC7 cells, and the ITGB4-DC vaccine plus anti–PD-L1-primed immune plasma killed ALDHhigh significantly more than theimmune plasma collected from all the other groups against ALDHhigh

cells (Fig. 5F). Together, these experiments demonstrated that ITGB4-targeted immunotherapies induced host immunity against ALDHhigh

CSCs as well as non-CSCs.

ITGB4-targeted immunotherapy reduced the proportion ofALDHhigh and ITGB4high cells in residual tumors and reduced thetumor-initiating capacity of these cells

The experiments described above suggested that ITGB4 immuno-therapy could target ALDHhigh CSCs that express this protein. Toconfirm this, we determined the proportion of ALDHhigh cells andITGB4high cells in residual tumors after treatment. After treatmentwith ITGB4-DC vaccine, the ALDHhigh population was reduced inboth 4T1 (from 6.74% to 1.11%; Fig. 6A) and SCC7 (from 3.11% to0.70%; Fig. 6B). This number was further decreased by cotreatmentwith anti–PD-L1 to 0.90% for 4T1 (Fig. 6A) and to 0.54% for SCC7(Fig. 6B). In parallel, the frequencies of ITGB4-expressing ALDHhigh

and ALDHlow 4T1 cells (Fig. 6C) and ALDHhigh and ALDHlow SCC7

Figure 5.

ITGB4-targeted immunotherapies elicited ITGB4-specific host immune responses against CSCs as well as non-CSCs. A and B, Splenetic T cells harvested frommicetreated with ITGB4-DC vaccine (A) or mITGB4 BiAb–armed TDLN T cells (B) mediated significant cytotoxicity against 4T1 cells. Data were replicated in a secondexperiment for both ITGB4-DC vaccine and mITGB4 BiAb–armed TDLN T-cell adoptive transfer experiments. C–F, ITGB4-targeted immunotherapy conferredsignificant host anti-ITGB4 humoral immunity in both 4T1 and SCC7models. Immune plasmawas collected from treatedmice, and IgGwas quantified using ELISA. C,Plasma harvested from treated mice-bearing 4T1 tumor, as indicated, bound to 4T1 cells. D, Plasma IgG induced by ITGB4-DC vaccine bound to unsorted, ALDHhigh,and ALDHlow SCC7 cells to a different extent. E, Cytotoxic effects of immune plasma via CDC in 4T1 model. Plasma harvested from animals bearing 4T1 tumorsubjected to treatmentwas incubatedwithALDHhigh 4T1 or ALDHlow 4T1 cells for 1 hour, followedby culturing in the presence of rabbit complement for another 1 hour.Lower percentageof viable cells at the endof incubation indicatesmore cell lysis.F, Similar CDCassayas inEwas tested in theSCC7model. Datawere repeated twice.

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cells (Fig. 6D) in the mITGB4-DC vaccine plus anti–PD-L1-treatedtumors were lower than those in monotherapy groups and PBScontrols.

We examined the change of ALDHhighITGB4high cells in thetreated residual tumors by double staining, and found that thepercentage of ALDHhighITGB4high cells was markedly reduced from7.83% and 10.6% in the control (PBS) group of 4T1 tumor (Fig. 6E)and SCC7 tumor (Fig. 6F) to 1.83% and 1.04% respectively aftercombination treatment with mITGB4-DC vaccine plus anti–PD-L1administration.

To assess if the immunotherapies also reduced the tumor-initiatingcapacity of 4T1 and SCC7 CSCs, we isolated ALDHhigh 4T1 (Fig. 6A)and SCC7 (Fig. 6B) cells from the residual tumors subjected tomITGB4-DC vaccine and/or anti–PD-L1 treatment and injected theminto normal syngeneic Balb/c and C3H mice respectively to test theirtumorigenicity. We found that although 50,000 ALDHhigh 4T1 cellsfrom PBS-treated control mice formed large tumors in 40 days, equal

number ofALDHhigh 4T1 cells frommITGB4-DCvaccination alone orcombination treatment with mITGB4-DC vaccination þ anti–PD-L1failed to form tumors within the same period of time (Fig. 7A and B).In the SCC7 model, 50,000 ALDHhigh SCC7 cells from mITGB4-DCvaccine� anti–PD-L1-treatedmice grew significantly slower than thatof other groups (Fig. 7C andD). Similar results were observed at lowernumbers (5,000) of ALDHhigh cells injected (Fig. 7C and E).

To confirm that ITGB4-targeted cancer immunotherapy has theability to reduce the tumorigenicity of the residual tumor cells, we alsotested the tumor-initiating capacity of the whole residual 4T1 andSCC7 cells.When 1� 106 residual 4T1 or SCC7 cells from treatedmicewere injected into both sides of syngeneic Balb/c and C3H micerespectively, equal number of the residual tumor cells from combinedmITGB4-DC vaccineþ anti–PD-L1-treatedmice formed significantlysmaller tumors than that from PBS or monotherapy-treated mice(Fig. 7F and G for 4T1; Fig. 7H and I for SCC7). Together, these datashow that ITGB4-targeted immunotherapy reduced both the number

Figure 6.

ITGB4-targeted immunotherapy reduced the number of ALDHhigh/ITGB4high CSCs. Flow cytometric proportions of ALDHhigh 4T1 (A) and SCC7 (B) cells in residualtumor subjected to treatment as indicated. Frequencies of ITGB4-expressing ALDHhigh and ALDHlow 4T1 (C) and SCC7 (D) cells from residual tumors treated asindicated. The coexpression of ALDH and ITGB4 in residual 4T1 tumors (E) and SCC7 tumors (F). The numbers of ALDHhigh/ITGB4high residual 4T1 (E) and SCC7 (F)cells were significantly reduced, particularly after combined treatment of DC vaccine plus anti–PD-L1. Results of flow cytometry are representative of two separateexperiments performed.

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of ALDHhigh and ITGB4high CSCs in residual 4T1 and SCC7 tumorsand their ability to form tumors, as well as suppressing the tumor-igenicity of the whole residual tumors subjected to ITGB4-targetedimmunotherapy.

Lack of systemic toxicity from ITGB4-targeted therapiesTo address the potential toxicity as well as off-target effects of

engaging ITGB4, we performed a group of experiments, including: (1)Histopathologic analysis of internal organs and glands from treated

Figure 7.

ITGB4-targeted immunotherapy reduced the tumorigenicity of CSCs and residual tumors. A–E, Tumor growth of residual ALDHhigh CSCs in syngeneic micewas significantly polarized in the 4T1 model (A and B) and in the SCC7model (C–E).A, 5� 104 residual ALDHhigh 4T1 cells were injected in both sides of Balb/c mice.C, The 5� 104 residual ALDHhigh SCC7 cells were injected in right side of C3Hmice (green arrows), whereas 5� 103 residual ALDHhigh SCC7 cells were injected to theleft side (yellow arrows). B, D, and E, Tumor growth curves resulted from injection of different doses of ALDHhigh tumor cells from treated residual tumors; 5 � 104

residual ALDHhigh 4T1 cells were injected into Balb/c mice (B), 5� 104 residual ALDHhigh SCC7 cells were injected into C3H mice (D), and 5� 103 residual ALDHhigh

SCC7 cellswere injected into C3Hmice (E).F–I, The tumorigenicity of thewhole residual tumor cellswas tested. F andH,Photos of representative tumors grown frominjected whole residual tumor cells subjected to treatment as indicated. Note that 1� 106 residual 4T1 (F) or SCC7 (H) cells were injected in both sides of syngeneicBalb/c and C3H mice, respectively. Tumor growth curves from whole residual 4T1 (G) and SCC7 (I) are shown.

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mice; (2) Hematologic analysis, and (3) Access of Insulin-like growthfactor-1 levels following treatment. As shown in Supplementary Fig. S1and Supplementary Table S1, these results as well as lack of weight lossin treated animals versus controls demonstrate the safety of thisapproach.

DiscussionIn this study, we investigated the immunologic targeting of ITGB4

utilizing two different approaches: mouse ITGB4 protein as antigen topulse DCs or coating TDLN T cells with mouse anti-CD3/anti-ITGB4BiAb to prepare mITGB4 BiAb-T cells for targeted adoptive transfer.Data obtained from applying two distinct immunologic methodsconfirmed that ITGB4 can serve as an effective target for cancerimmunotherapies.

4T1 cells highly expressed ITGB4 in unsorted, ALDHhigh, andALDHlow cells. The 4T1 mammary carcinoma is highly tumorigenicand invasive and can spontaneously metastasize from the primarytumor in the mammary gland to multiple distant sites includinglymph nodes, blood, liver, lung, brain, and bone (43). In the 4T1minimal tumor model, mITGB4-DC vaccine inhibited both localtumor growth and spontaneous pulmonary metastases. In addition,mITGB4 BiAb–armed T-cell adoptive transfer suppressed tumorgrowth of established macroscopic 4T1 tumor. In an experimentallung metastasis model, BiAb-armed T-cell adoptive transfer alsoinhibited lung metastases. Together, these data indicate that bothmITGB4-DC vaccine and mITGB4 BiAb–armed T-cell adoptivetransfer can suppress local tumor growth and metastasis of a tumorwith high expression of ITGB4 on both ALDHhigh CSCs and bulktumor cells.

In contrast, SCC7 cells expressed ITGB4 on the ALDHhigh cellssignificantly more than that on the ALDHlow cells. mITGB4-DCvaccine and mITGB4 BiAb–armed T-cell adoptive transfer effectivelysuppressed the local tumor growth as well as the experimentalmetastasis of SCC7, suggesting that successful immunologic targetingof ITGB4 is able to target ITGB4-expressing CSCs that mediate tumormetastasis, as well as ITGB4-expressing bulk tumor cells.

By using 4T1-ITGB4KO cells and CT26 cells lacking ITGB4expression as negative controls, our data confirmed the specificityof ITGB4 targeting. Interestingly, knockout of ITGB4 significantlyreduced the tumor growth of 4T1 cells. This suggests that ITGB4plays an important biological role and supports a recent report thatknockout of ITGB4 reduced the migration and invasion of HCT116colorectal cancer cells (44). Together, these data strongly supportthe conclusion that ITGB4 represents an attractive target for cancerimmunotherapy.

We also examined the ability of ITGB4 immunotherapy to augmentthe efficacy of anti–PD-L1 immune checkpoint blockade becausePD-L1 was expressed in our tumor cell models. Furthermore, Wangand colleagues reported that PD-L1 can promote the growth andmetastasis of tumor by activating the ITGB4/SNAI1/SIRT3 signalingpathway, suggesting another potential mechanism of synergy betweenthese pathways (45). Our data indicate that anti–PD-L1 antibodysignificantly augmented the therapeutic efficacy of ITGB4-DC vaccineand ITGB4 BiAb–armed T-cell adoptive transfer in both 4T1 andSCC7 models.

We found in this study that ITGB4-targeted immunotherapiescould elicit ITGB4-specific cellular immune responses. CTLs gener-ated from the spleens of treated 4T1-bearing mice specifically killed

ITGB4-expressing 4T1 tumor cells. In addition, ITGB4-DC vaccineconferred significant host anti-ITGB4 humoral immunity. ITGB4-DCvaccine–primed immune plasma specifically bound to 4T1 and SCC7cells, resulting in significant killing of ITGB4-expressing 4T1 andSCC7 cells. Toxicity remains a major concern for any kind of immu-notherapy. ITGB4 is expressed in the skin, and its expression corre-lated with epidermal lysis bullosa (46). However, in this study we didnot detect systemic toxicity associated with ITGB4 immunotherapy inour mouse models.

In both approaches of ITGB4-targeted immunotherapy, weobserved a significantly reduced number of ITGB4-expressing4T1 cells and SCC7 cells. In parallel, immunotherapeutic targetingof ITGB4 significantly reduced the ALDHhigh 4T1 cells and ALDH-high SCC7 CSCs in the residual tumors subjected to immunotherapy.Importantly, the residual ALDHhigh 4T1 cells and SCC7 CSCsdemonstrated significantly reduced tumorigenicity. In addition,significantly reduced tumorigenicity of whole residual tumors sub-jected to ITGB4-targeted immunotherapy confirmed that ITGB4-targeted immunotherapy could reduce both the number and thetumorigenicity of the residual 4T1 and SCC7 tumors. BecauseITGB4 is expressed in CSCs across a number of tumor types,immunologic targeting of this protein has significant therapeuticpotential.

Disclosure of Potential Conflicts of InterestL. Lum is SAB member at Rapa Therapeutics, reports receiving from com-

mercial research grant from Dendreon and Pharmacyclics, and has an ownershipinterest (including patents) in Transtarget, Inc. E.M. Hurt is Principal Scientistat, and has an ownership interest (including patents) in, AstraZeneca. M.S. Wichahas an ownership interest (including patents) in Oncomed Pharmaceuticalsand has an unpaid consultant/advisory board relationship with MedImmune.Q. Li had a consultant relationship with Guangzhou Improve Medical Instru-ments Co., Ltd. Y. Zhu is Principal Scientist at Guangzhou Improve MedicalInstruments Co., Ltd. No potential conflicts of interest were disclosed by the otherauthors.

Authors’ ContributionsConception and design: L. Lum, E.M. Hurt, M.S. Wicha, Q. LiDevelopment of methodology: S. Ruan, L. Lum, A. Thakur, M.S. WichaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): S. Ruan, M. Lin, W. Shao, Y. Zhang, Y. HuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ruan, A. Thakur, A.E. Chang, M.S. WichaWriting, review, and/or revision of the manuscript: S. Ruan, L. Lum, A. Thakur,A.E. Chang, M.S. Wicha, Q. LiAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Y. Zhu, E.M. Hurt, M.S. WichaStudy supervision: R. Jin, S. Huang, E.M. Hurt, Q. Li

AcknowledgmentsThis work was partially supported by Medimmune Inc. (M.S. Wicha and Q. Li),

Guangzhou Improve Medical Instruments Co., Ltd. (Q. Li), Gillson LongenbaughFoundation (A.E. Chang and Q. Li), R01 CA 140314 (L. Lum), R01 CA 182526 (L.Lum), and the University of MichiganMICHRGrant UL1TR000433 (Q. Li), the NCIresearch grant 1R-35CA 197585 (M.S. Wicha), as well as the NCI research grantP30CA046592 (T. Moore).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 9, 2019; revised September 30, 2019; accepted December 10, 2019;published first December 16, 2019.

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References1. Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: the mechanistic link

and clinical implications. Nat Rev Clin Oncol 2017;14:611–29.2. Eun K, Ham SW, Kim H. Cancer stem cell heterogeneity: origin and new

perspectives on CSC targeting. BMB Reports 2017;50:117–25.3. Dawood S, Austin L, Cristofanilli M. Cancer stem cells: implications for cancer

therapy. Oncology (Williston Park) 2014;28:1101–7, 1110.4. Feng D,Wang N, Hu J, LiW. Surface markers of hepatocellular cancer stem cells

and their clinical potential. Neoplasma 2014;61:505–13.5. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110:

673–87.6. Kajiji S, Tamura RN, Quaranta V. A novel integrin (alpha E beta 4) from human

epithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J1989;8:673–80.

7. Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications andtherapeutic opportunities. Nat Rev Cancer 2010;10:9–22.

8. Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G, et al. Beta 4integrin amplifies ErbB2 signaling to promote mammary tumorigenesis. Cell2006;126:489–502.

9. Muthuswamy SK. ErbB2 makes beta 4 integrin an accomplice in tumorigenesis.Cell 2006;126:443–5.

10. Lee WJ, Chen WK, Wang CJ, Lin WL, Tseng TH. Apigenin inhibits HGF-promoted invasive growth andmetastasis involving blocking PI3K/Akt pathwayand beta 4 integrin function in MDA-MB-231 breast cancer cells. Toxicol ApplPharmacol 2008;226:178–91.

11. Muranen T, Iwanicki MP, Curry NL, Hwang J, DuBois CD, Coloff JL, et al.Starved epithelial cells uptake extracellular matrix for survival. Nat Commun2017;8:13989.

12. Lee JG, Ahn JH, JinKT,Ho LJ, Choi JH.Mutant p53 promotes ovarian cancer celladhesion to mesothelial cells via integrin beta4 and Akt signals. Sci Rep 2015;5:12642.

13. Leng C, Zhang ZG, ChenWX, Luo HP, Song J, DongW, et al. An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancinganchorage independence through activation of FAK-AKT pathway. Cancer Lett2016;376:188–96.

14. Ephstein Y, Singleton PA, ChenW,Wang L, Salgia R, Kanteti P, et al. Critical roleof S1PR1 and integrin beta4 in HGF/c-Met-mediated increases in vascularintegrity. J Biol Chem 2013;288:2191–200.

15. Tai YL, Lai IR, Peng YJ, Ding ST, Shen TL. Activation of focal adhesion kinasethrough an interactionwith beta4 integrin contributes to tumorigenicity of coloncancer. FEBS Lett 2016;590:1826–37.

16. Cooper J, Giancotti FG. Integrin signaling in cancer: mechanotransduction,stemness, epithelial plasticity, and therapeutic resistance. Cancer Cell 2019;35:347–67.

17. Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic MM, et al.Tumour exosome integrins determine organotropic metastasis. Nature 2015;527:329–35.

18. Kawakami K, Fujita Y, Kato T, Mizutani K, Kameyama K, Tsumoto H, et al.Integrin beta4 and vinculin contained in exosomes are potential markers forprogression of prostate cancer associated with taxane-resistance. Int J Oncol2015;47:384–90.

19. Masugi Y, Yamazaki K, Emoto K, Effendi K, Tsujikawa H, Kitago M, et al.Upregulation of integrin beta4 promotes epithelial-mesenchymal transition andis a novel prognostic marker in pancreatic ductal adenocarcinoma. Lab Invest2015;95:308–19.

20. Brendle A, Lei H, Brandt A, Johansson R, Enquist K, Henriksson R, et al.Polymorphisms in predicted microRNA-binding sites in integrin genes andbreast cancer: ITGB4 as prognostic marker. Carcinogenesis 2008;29:1394–9.

21. Yoshioka T, Otero J, Chen Y, Kim YM, Koutcher JA, Satagopan J, et al. beta4Integrin signaling induces expansion of prostate tumor progenitors. J Clin Invest2013;123:682–99.

22. Bierie B, Pierce SE, Kroeger C, Stover DG, Pattabiraman DR, Thiru P, et al.Integrin-beta4 identifies cancer stem cell-enriched populations of partiallymesenchymal carcinoma cells. Proc Natl Acad Sci U S A 2017;114:E2337–46.

23. KrogerC,AfeyanA,Mraz J, Eaton EN,Reinhardt F, KhodorYL, et al. Acquisitionof a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells.Proc Natl Acad Sci U S A 2019;116:7353–62.

24. PeitzschC,TyutyunnykovaA, Pantel K,DubrovskaA. Cancer stem cells: the rootof tumor recurrence and metastases. Semin Cancer Biol 2017;44:10–24.

25. Chang JC. Cancer stem cells: role in tumor growth, recurrence, metastasis, andtreatment resistance. Medicine (Baltimore) 2016;95(1 Suppl 1):S20–5.

26. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, BrownM, et al. ALDH1 is a marker of normal and malignant human mammarystem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007;1:555–67.

27. Prince M, Zhou L, Moyer JS, Tao H, Lu L, Owen J, et al. Evaluation of theimmunogenicity of ALDH(high) human head and neck squamous cell carci-noma cancer stem cells in vitro. Oral Oncol 2016;59:30–42.

28. NingN, PanQ, Zheng F, Teitz-Tennenbaum S, EgentiM, Yet J, et al. Cancer stemcell vaccination confers significant antitumor immunity. Cancer Res 2012;72:1853–64.

29. Lu L, Tao H, Chang AE, Hu Y, Shu G, Chen Q, et al. Cancer stem cell vaccineinhibits metastases of primary tumors and induces humoral immune responsesagainst cancer stem cells. OncoImmunology 2015;4:e990767.

30. Hu Y, Lu L, Xia Y, Chen X, Chang AE, Hollingsworth RE, et al. Therapeuticefficacy of cancer stem cell vaccines in the adjuvant setting. Cancer Res 2016;76:4661–72.

31. Lum LG, Thakur A. Targeting T cells with bispecific antibodies for cancertherapy. BioDrugs 2011;25:365–79.

32. Thakur A, Rathore R, Kondadasula SV, Uberti JP, Ratanatharathorn V, Lum LG.Immune T cells can transfer and boost anti-breast cancer immunity. OncoIm-munology 2018;7:e1500672.

33. Lum LG, Thakur A, Al-Kadhimi Z, Colvin GA, Cummings FJ, Legare RD, et al.Targeted T-cell therapy in stage IV breast cancer: a phase I clinical trial.Clin Cancer Res 2015;21:2305–14.

34. Huang J, Li C, Wang Y, Lv H, Guo Y, Dai H, et al. Cytokine-induced killer(CIK) cells bound with anti-CD3/anti-CD133 bispecific antibodies targetCD133(high) cancer stem cells in vitro and in vivo. Clin Immunol 2013;149:156–68.

35. Li Q, Lao X, PanQ, NingN, Yet J, Xu Y, et al. Adoptive transfer of tumor reactiveB cells confers host T-cell immunity and tumor regression. ClinCancer Res 2011;17:4987–95.

36. Brattain MG, Strobel-Stevens J, Fine D, Webb M, Sarrif AM. Establishment ofmouse colonic carcinoma cell lines with different metastatic properties.Cancer Res 1980;40:2142–6.

37. Li Q, Lu L, Tao H, Xue C, Teitz-Tennenbaum S, Owen JH, et al. Generation of anovel dendritic-cell vaccine using melanoma and squamous cancer stem cells.J Vis Exp 2014;e50561.

38. LiQ, CarrA, Ito F, Teitz-TennenbaumS,ChangAE. Polarization effects of 4-1BBduring CD28 costimulation in generating tumor-reactive T cells for cancerimmunotherapy. Cancer Res 2003;63:2546–52.

39. Thakur A, Lum LG, Mittal S. Bispecific antibody armed T cells to target cancercells. Methods Mol Biol 2018;1722:117–26.

40. Lou Y, McDonald PC, Oloumi A, Chia S, Ostlund C, Ahmadi A, et al. Targetingtumor hypoxia: suppression of breast tumor growth and metastasis by novelcarbonic anhydrase IX inhibitors. Cancer Res 2011;71:3364–76.

41. Yang S, Zhang JJ, Huang XY. Mouse models for tumor metastasis. Methods MolBiol 2012;928:221–8.

42. Price JE. Spontaneous and experimental metastasis models: nude mice.Methods Mol Biol 2014;1070:223–33.

43. Pulaski BA, Ostrand-Rosenberg S. Mouse 4T1 breast tumor model. Curr ProtocImmunol 2001;Chapter20:20–2.

44. Choi S, MarcoM, Chen C, Pelossof R, O'Rourke K, Smith J, et al. KRASmutationis associated with upregulation of integrin beta-4 expression leading to tumorinvasion in colorectal cancer. J Clin Oncol; 2019;37;576.

45. Wang S, Li J, Xie J, Liu F, Duan Y, Wu Y, et al. Programmed death ligand 1promotes lymph node metastasis and glucose metabolism in cervical cancer byactivating integrin beta4/SNAI1/SIRT3 signaling pathway. Oncogene 2018;37:4164–80.

46. Nakano A, Pulkkinen L, Murrell D, Rico J, Lucky AW, Garzon M, et al.Epidermolysis bullosa with congenital pyloric atresia: novel mutations in thebeta 4 integrin gene (ITGB4) and genotype/phenotype correlations. Pediatr Res2001;49:618–26.

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Immunologic Targeting of Integrin b4

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2020;80:771-783. Published OnlineFirst December 16, 2019.Cancer Res   Shasha Ruan, Ming Lin, Yong Zhu, et al.   Growth and Decrease Metastasis

Targeted Cancer Immunotherapies Inhibit Tumor−4βIntegrin

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