Tumor-Promoting Desmoplasia Is Disrupted by Depleting FAP ... · cell proliferation, survival, invasion, and epithelial-to-mesenchy-mal transition (EMT; refs. 29 and 30). Genetic

Microenvironment and Immunology

Tumor-Promoting Desmoplasia Is Disrupted byDepleting FAP-Expressing Stromal CellsAlbert Lo1,2, Liang-Chuan S.Wang3, John Scholler4, James Monslow1, Diana Avery1,Kheng Newick3, Shaun O'Brien3, Rebecca A. Evans2,4, David J. Bajor4,5,Cynthia Clendenin4, Amy C. Durham6, Elizabeth L. Buza6, Robert H. Vonderheide2,4,5,Carl H. June2,4,7, Steven M. Albelda2,3,5, and Ellen Pur�e1,2,4

Abstract

Malignant cells drive the generation of a desmoplastic andimmunosuppressive tumor microenvironment. Cancer-associat-ed stromal cells (CASC) are a heterogeneous population thatprovides both negative and positive signals for tumor cell growthandmetastasis. Fibroblast activationprotein (FAP) is amarker of amajor subset of CASCs in virtually all carcinomas. Clinically, FAPexpression serves as an independent negative prognostic factor formultiple types of human malignancies. Prior studies establishedthat depletion of FAPþ cells inhibits tumor growth by augmentingantitumor immunity. However, the potential for immune-inde-pendent effects on tumor growth have not been defined. Herein,we demonstrate that FAPþCASCs are required formaintenance of

the provisional tumor stroma because depletion of these cells, byadoptive transfer of FAP-targeted chimeric antigen receptor (CAR)T cells, reduced extracellular matrix proteins and glycosaminogly-cans. Adoptive transfer of FAP-CAR T cells also decreased tumorvascular density and restrained growth of desmoplastic humanlung cancer xenografts and syngeneicmurine pancreatic cancers inan immune-independent fashion. Adoptive transfer of FAP-CARTcells also restrained autochthonous pancreatic cancer growth.These data distinguish the function of FAPþ CASCs from otherCASC subsets and provide support for further development ofFAPþ stromal cell-targeted therapies for the treatment of solidtumors. Cancer Res; 75(14); 2800–10. �2015 AACR.

IntroductionCarcinomas are complex tumors consisting of neoplastic epi-

thelial cells, vasculature, inflammatory cells, and immune cellinfiltrates. Many human carcinomas exhibit desmoplasia, char-acterizedby the accretionof reactive stromal cells and extracellularmatrix (ECM). In established human solid tumors, nests of cancercells are often circumscribed by a dense fibrotic stroma containinghigh levels of collagen, fibronectin, and hyaluronan (HA), and aheterogeneous population of cancer-associated stromal cells(CASC), including cancer-associated fibroblasts (CAF), a smoothmuscle actin-positive (aSMAþ) myofibroblasts, and mesenchy-

mal stem cells (MSC; refs. 1–5). The extent of desmoplasia variesamong different tumor types. In pancreatic cancer, desmoplasiacomprises as much as 90% of tumor mass and heightens thera-peutic resistance (6). However, even in tumors in which stromarepresents a relativelyminor component, desmoplasia can impacttumor cell behavior. The role of desmoplasia in tumor initiation,progression, metastasis, and resistance to therapy is complex andnot yet well understood.

Thedesmoplastic responsecanpromote tumorgrowth, invasion,andmetastasis through ECM remodeling as well as the productionof growth factors, cytokines, and chemokines (7–10). It alsopromotes tumorigenesis by supporting angiogenesis (7, 11), alter-ing tissue stiffness and mechanotransduction (12, 13), inducinginflammation (14, 15), and suppressing antitumor immunity(16, 17). Tumor stroma can also limit drug delivery and conferresistance to chemotherapeutics (18–21), radiation (22), anti-angiogenesis therapy (23), and immunotherapy (16, 24, 25).

Based on the tumor-promoting functions and the therapeuticresistance conferred by tumor stroma, it has been hypothesizedthat destruction of stromal cells and/or disruption of molecularstromal cell/ECM-dependent pathways would inhibit tumorgrowth and augment efficacy of other therapeutic modalities.Several proof-of-concept studies support this hypothesis. Prevent-ing recruitment and differentiation of CASCs by targetingchemokine–chemokine receptor pathways inhibited tumor pro-gression (26, 27). Consistent with the role of inflammatorymyeloid cells in promoting CAF activation, treatment with dexa-methasone reduced desmoplasia and attenuated tumor growth(15). Furthermore, inhibition of collagen binding to its discoidindomain receptor reduced colony formation of primary pancreatictumor cells (28). Blockade of the HA-CD44 axis inhibited tumor

1Department of Biomedical Sciences, University of Pennsylvania, Phil-adelphia, Pennsylvania. 2Cell and Molecular Biology Graduate Group,University of Pennsylvania, Philadelphia, Pennsylvania. 3ThoracicOncology Research Laboratory, University of Pennsylvania, Philadel-phia, Pennsylvania. 4Abramson Family Cancer Research Institute,University of Pennsylvania, Philadelphia, Pennsylvania. 5Departmentof Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.6Department of Pathobiology, School of Veterinary Medicine, Univer-sity of Pennsylvania, Philadelphia, Pennsylvania. 7Department ofPathology and Laboratory Medicine, Perelman School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania.

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

A. Lo, L.-C. Wang, S.M. Albelda, and E. Pur�e contributed equally to this article.

Corresponding Author: Ellen Pur�e, Hill Pavilion Room 410E, 380 S. UniversityAvenue, Philadelphia, PA 19104. Phone: 215-573-9406; Fax: 215-573-6810;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-3041

�2015 American Association for Cancer Research.

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cell proliferation, survival, invasion, and epithelial-to-mesenchy-mal transition (EMT; refs. 29 and 30). Genetic targeting orpharmacological inhibition of the protease activity of fibroblastactivation protein (FAP) reduced lung tumor growth (9). Further-more, depletion of HA in the tumor stroma induced a transientincrease in vessel density and perfusion that facilitated delivery ofgemcitabine into tumors, thereby augmenting efficacy of chemo-therapy in a highly desmoplastic mouse model of pancreaticductal adenocarcinoma (PDA; refs. 18 and 20).

An alternative approach to targeting molecular pathways is totarget stroma at a cellular level; however, it is important to keep inmind the potential plasticity (31, 32) and heterogeneity of thiscompartment (1, 3). Distinct stromal cell subpopulations mayhave opposing effects on tumor growth, progression, and metas-tasis. The depletion of specific subpopulations may have eithertherapeutic or detrimental effects. The impact may depend ontumor type, stage of tumor progression, variation in tumorimmunogenicity, and the degree of desmoplasia. Thus, delinea-tion of the roles of distinct subpopulations in tumor growth,including their roles in regulating antitumor immunity, desmo-plasia, and angiogenesis, is required to inform the rational designof stromal cell–targeted therapies.

For many years, SMAþ myofibroblasts have been noted in avariety of solid tumors. However, recent analyses have revealedadditional phenotypically distinct subsets of CASCs. Notably,FAP is expressed on the majority of activated fibroblasts andMSCs found in the tumor microenvironment, only a subset ofwhich co-express SMA (1, 3). Interestingly, studies also indicatethat a lineage relationship may exist between MSC progenitorsand terminally differentiated SMAþ myofibroblasts (27, 33).Specific ablation of SMAþ myofibroblasts in mouse models ofPDA suppressed antitumor immunity, enhanced hypoxia andEMT, and reduced survival (34). Notably, the approach used inthis study to deplete SMAþ cells did not impact the prevalence ofFAPþ CASCs and had a modest and selective effect on ECM. Incontrast to the impact of depleting SMAþmyofibroblasts, targeteddeletion of FAPþ cells using genetic (16, 35–37) and immune-based approaches (38–44) inhibited tumor growth. Thus, SMAþ

and FAPþ stromal cells may differentially regulate tumorigenesis.Although our mechanistic understanding of the impact of

depleting CASCs is still limited, several studies demonstratedthat depleting FAPþ CASCs enhanced antitumor immunity(16, 35, 36, 44). We found that depleting FAPþ cells in murineAE17.OVA mesothelioma or TC-1 lung tumors increased intra-tumoral T cells and that the antitumor effect of depleting FAPþ

cells was abrogated in immune-deficient hosts, indicating anessential role for adaptive immunity. However, the tumormodelsused in that study were highly immunogenic and exhibitedmodest desmoplasia. Nevertheless, we detected pronouncedalterations in stromal composition. These studies therefore raisedthe question as towhether depletion of FAPþ cells in the setting ofweakly immunogenic and/or highly desmoplastic tumors mayaffect tumor growth through immune-independent stromal-dependent mechanisms. In the study described herein, we eval-uated the requirement of FAPþ cells for the maintenance of adesmoplastic stroma and the impact of FAP-CAR T-cell–mediateddepletion of FAPþ cells on weakly immunogenic, but highlydesmoplastic tumors under conditions in which immune andstromal-dependent mechanisms could be dissected. We demon-strate that FAPþ cells are required tomaintain a provisionalmatrixin desmoplastic tumors, including human lung cancer andmeso-

thelioma xenografts and mouse models of PDA, and the efficacyof stromal cell depletion in inhibiting tumor growth in suchtumors ismediated by immune-independentmechanisms. Takentogether with recent data from others (16, 35, 36), these resultsdefine potentially distinct roles for myofibroblasts and FAPþ

stromal cells in pancreatic cancer (34).

Materials and MethodsCell culture

Mouse AE17 mesothelioma cells expressing chicken ovalbu-min (AE17.OVA) were provided by Dr. Delia Nelson (Universityof Western Australia, Crawley, WA, Australia). Murine 4662 PDAcells were derived from a pancreatic tumor isolated from a fullybackcrossed C57BL/6 KrasG12D:Trp53R172H:Pdx-1-Cre (KPC)mouse, recapitulating the desmoplastic feature of autochthonousPDA (45). Human A549 lung adenocarcinoma cells were pur-chased from the American Type Culture Collection. Humansarcomatoid type mesothelioma cells I45 were provided byDr. J. Testa (Fox Chase Institute, Philadelphia, PA). Cells wereauthenticated by morphology, growth characteristics and bio-logic behavior, tested mycoplasma-free and frozen. Cells werecultured less than 1 month after resuscitation.

AnimalsC57BL/6 mice and NOD/SCID/IL2-receptor g chain knockout

(NSG)mice were purchased from Charles River Laboratories Inc.,Jackson Labs, and the Children's Hospital of Philadelphia. FAP-null mice (FapLacZ/LacZ) provided by Boehringer Ingelheim (46)were backcrossed 12 generations onto a C57BL/6 genetic back-ground. These mice in turn were crossed with NSG mice togenerate NSG FAP-null mice. KPC mice were provided by mousehospital at the University of Pennsylvania. Experimental proto-cols were approved by the Institutional Animal Care andUse Committee and were in compliance with the Guideline forthe Care and Use of Animals.

Engineering of anti-muFAP CAR constructs and generation ofFAP-CAR T cells

A single-chain Fv domain of anti-mouse FAP antibody 73.3alongwith humanCD8ahinge and transmembrane domain, plus4-1BB and CD3z signaling domains were cloned into the retro-viral vectorMigR1 encoding EGFP to establish FAP-CAR constructas described (44). Primary mouse T cells were transduced withFAP-CAR construct or MigR1 vector to generate FAP-CAR T cellsand MigR1 T cells respectively, as described in SupplementaryMaterials and Methods.

Intravenous transfer of CAR-T cells in mice bearing establishedtumors

C57BL/6 mice and NSG mice were injected subcutaneouslywith 2� 106 AE17.OVAor 3� 105 4662 tumor cells suspended inmatrigel (BD Biosciences). Tumor-bearing mice were randomlyassigned to three groups that received FAP-CAR or MigR1-trans-duced (control) mouse T cells or remained untreated. KPC micewere monitored by ultrasound and randomly assigned for treat-ment when they had established pancreatic tumors of 95 to 270mm3.

A total of 1 � 107 CAR T cells per dose were administeredintravenously. T cells were given once for AE17.OVA tumor-bearing mice and twice (at a week interval) for PDA models. Totest human T cells with redirected CAR against murine FAP in

FAPþ Cells Are Required for Tumor-Promoting Desmoplasia

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human tumor xenografts, NSG and NSG-FAP null mice wereinjected subcutaneously with 2 � 106 A549 cells or I45 cellsmixed with matrigel. Mice bearing established tumors thenreceived one dose of 1 � 107 FAP-CAR human T cells intrave-nously or left untreated.

Statistical analysesFor studies comparing two groups, the Student t test was used.

For comparisons of more than two groups, we used one-wayANOVAwith appropriate post hoc testing. Statisticswere calculatedusing GraphPad Prism 5. Data are presented as mean � SEM.Differences were considered significant when P < 0.05.

ResultsThe extent of desmoplasia in mouse tumors and human tumorxenografts correlates with the preponderance of FAPþ cells

We examined the extent of desmoplasia in several mousetumors and human tumor xenografts. The murine pancreatic

cancer cell line 4662 transplanted into syngeneic C57BL/6 miceand A549 human lung adenocarcinoma cells in NSG mice werecircumscribed by dense stroma. In contrast, AE17.OVA mousemesothelioma and I45 human mesothelioma tumors exhibitedmoderate and sparse desmoplasia, respectively (Fig. 1A). Exten-sive accumulation of collagen and glycosaminoglycans (GAG)was evident in 4662 and A549 tumors compared with in AE17.OVA and I45 tumors (Fig. 1A). In particular, high levels of HA,known to regulate tumor cell behavior (29, 30) and conferresistance to chemotherapy (18, 20, 21, 30), were detected onlyin the more desmoplastic tumors. The levels and spatial distri-bution of HA correlated with those of versican, a proteoglycanimplicated in tumor progression that associateswithHA (Fig. 1A).Notably, all matrix components examined were dispersed dif-fusely throughout the AE17.OVA and I45 tumors compared withthe restricted localization of highly concentrated matrix compo-nents in the stromal regions of desmoplastic 4662 and A549tumors (Fig. 1A).

Figure 1.Extent of the desmoplastic stromalresponse in mouse and human tumorscorrelates with the prevalence ofintratumoral FAPþ stromal cells. A, theextent of desmoplasia was evaluatedin established tumors by hematoxylinand eosin (H&E) staining, Masson'strichrome staining to reveal collagendeposition (blue), Alcian blue stainingfor GAGs (aqua blue), and reactivityfor hyaluronan-binding protein(HABP) or anti-versican antibody.Scale, 100 mm. B, flow cytometry wasperformed to identify CD90þ stromalcells and CD45þ hematopoietic cellsthat express FAP. Data shown arenormalized to total live tumorpopulation andexpressed as themean� SEM (n > 5). �, significant differencebetween each group, P < 0.01.

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We next investigated the relationship between the extent ofdesmoplasia and the preponderance of FAPþ CASCs. In A549tumors, the majority of FAPþ CASCs were CD45�CD90þ stromalcells, although we consistently detected a minor population ofFAPþCASCs that expressed the hematopoietic cellmarker CD45þ

(Supplementary Fig. S1A), further characterized as F4/80þCD206þ (M2-like) macrophages (Supplementary Fig. S1B).Interestingly, the proportion of CD45�CD90þFAPþ stromal cellsin the various tumor types correlated with the degree of desmo-plasia. FAPþ stromal cells represented 8%and12%of total cells inthe highly desmoplastic 4662 and A549 tumors, respectively,compared with only 0.3% to 1.2% in AE17.OVA tumors andI45 xenografts (Fig. 1B). The level of FAPþ M2-like macrophages(CD45þF4/80þCD206þ) also correlated with the degree of des-moplasia, representing 0.34% and 1.65% in 4662 and A549tumors, respectively, compared with just 0.1% of total cells inAE17.OVA tumors and I45 xenografts (Fig. 1B). These data raisedthe possibility that FAPþ cells may be required for the formationand/or maintenance of a desmoplastic stroma.

We previously reported that adoptively transferred FAP-CAR Tcells infiltrated tumors, depleted FAPhi CASCs and inhibitedgrowth of AE17.OVA tumors (Supplementary Fig. S2A; ref. 44).The impact on tumor growth in this model depended on antitu-mor immunity, as the effect of FAP-CAR T cells was abrogated inimmune-deficientmice (Supplementary Fig. S2B; ref. 44). Furtheranalysis of tumors from FAP-CAR T-cell–treated mice revealedaltered tumor morphology and nuclear condensation (Supple-mentary Fig. S2C), reduced tumor cell proliferation (Supplemen-tary Fig. S3A), and increased tumor cell apoptosis (SupplementaryFig. S3B) compared with tumors from mice treated with controlMigR1 T cells. Stroma was also remarkably altered in FAP-CAR T-

cell–treated tumors. Collagen and HA were reduced by 57% and83% (P < 0.05), respectively (Supplementary Figs. S3C and S3D),and the number of CD31þ vessels was decreased by 30% (P <0.05) after FAP-CAR T cell infusion (Supplementary Fig. S3E). Incontrast, the stroma in tumors of mice that received MigR1 T cellswas similar to that in the untreated control (Supplementary Figs.S3C–S3E). These data establish that FAPþ stromal cells arerequired for the maintenance of desmoplasia. These dataprompted us to investigate whether FAPþ CASCs might promotetumor growth through immune-independent stromal-dependentmechanisms.

FAP-CAR T cells can mediate immune-independent growthinhibition of desmoplastic human xenografts

To address whether FAPþ CASC-dependent desmoplasia couldpromote tumor growth independently of suppressing adaptiveanti-tumor immunity, we assessed the impact of FAP-CAR T cellson the growth of established A549 human tumor xenografts inNSG mice. Administration of FAP-CAR human T cells directedagainst mouse FAP significantly reduced the growth of A549tumors with a 42% (P < 0.01) reduction in tumor size at day 8post-T cell transfer (Fig. 2A). Treatment with FAP-CAR T cellssignificantly reduced tumor cell proliferation and induced apo-ptosis inA549 tumors (Fig. 2DandE). In contrast, FAP-CART cellshad no impact on the growth of A549 tumors in FAP-null NSGmice (Fig. 2B), indicating that FAPexpressionbyhost stromal cellsis required for the anti-tumor effect elicited by FAP-CAR T cells.On the other hand, FAP-CAR T cells had no effect on human I45tumor xenografts (Fig. 2C), which induced little if any desmo-plastic response, indicating that the anti-tumor activity is depen-dent on the presence of intra-tumoral FAPþ cells and not likely

Figure 2.FAP-CAR T cells confer antitumoractivity in desmoplastic human lungcancer in immunodeficient hosts. A,NSG mice bearing established A549tumors received FAP-CAR human Tcells intravenously. B, to test targetspecificity of FAP-CAR T cells, NSGFAP-null tumor-bearing mice wereinjected intravenously with FAP-CARhuman T cells. C, NSG mice-bearingI45 tumors were injectedintravenously with FAP-CAR humanT cells. The black arrow indicates thetime of T-cell transfer (n ¼ 5 pergroup). A549 tumors were harvested8 days postadoptive T-cell transfer.Tumor sections were stained withantibodies against human-specificKi-67 (D) and cleaved caspase-3(E; n ¼ 3 per group). � , statisticalsignificance between untreated andFAP-CAR T-cell–treated samples,P < 0.01. Scale, 100 mm.






due to an indirect effect through off-tumor targeting of FAPþ cellsin normal tissues. Further studies demonstrated that FAP-CAR Tcells inhibited growth of two other highly desmoplastic humanmesothelioma xenografts in NSG mice (data not shown). Thus,immune-independent FAP-CAR T cell-mediated inhibition oftumor growth may depend on the degree of stromagenesis.

FAP-CAR T cells disrupt stromagenesis, angiogenesis, and theductal-like structure of tumor nodules in highly desmoplastichuman lung cancer xenografts

To investigate themechanisms by which FAPþCASC depletionleads to immune-independent growth inhibition of highly des-moplastic human tumor xenografts,we analyzed the compositionand architecture of A549 tumors 8 days post–T-cell transfer.Treatmentwith human FAP-CART cells in A549 tumors decreasedFAPþ CASCs by 84% (P < 0.01; Fig. 3A). Furthermore, in spite ofonly a minor population of FAPþ cells coexpressing SMA (Sup-plementary Fig. S1C), we also observed an 83% decrease (P <0.01) in SMAþmyofibroblasts (Fig. 3B), suggesting that SMAþ cellare either derived from progenitor populations, such as a sub-population of FAPþ MSCs that express CD105 and CD44 (Sup-plementary Fig. S1D), or that FAPþ cells are required for therecruitment or differentiation of SMAþ myofibroblasts. Interest-ingly, we observed a marked disruption of the adenocarcinomaductal-like structure of the tumor nodules and an increase innecrosis in FAP-CAR T-cell–treated tumors (Supplementary Fig.S4). These data indicate that extrinsic signals provided by stromalcells and/or ECM are required to maintain the ductal-like spatialorganization of tumor cells.

As angiogenesis is critical for tumor progression and is knownto be dependent on stromal cells and matrix (7, 11, 23), we

investigated whether FAP-CAR T cells affected vascularization ofA549 tumor xenografts. Quantification of CD31þ (endothelialcell) and NG2þ (pericyte) staining indicated that treatment withFAP-CAR T cells resulted in a 53% decrease (P < 0.01) in tumorendothelial cells (Fig. 3C) and 92% decrease (P < 0.01) inpericytes (Fig. 3D) when compared with tumors from untreatedmice.

FAP-CAR T cells resolve desmoplasia in highly desmoplastichuman lung cancer xenografts

Because the preponderance of FAPþ cells correlated with thedegree of desmoplasia, we investigated whether FAPþ cells arerequired to maintain desmoplasia in well-established tumors.Collagen content was decreased by 72% (P < 0.01; Fig. 4A) andfibronectin by 55% (P < 0.05; Fig. 4B) in FAP-CAR T cell-treatedA549 tumors. Total GAGs and HA were reduced by 57% (P <0.05) and 75% (P < 0.01), respectively (Fig. 4C and D), in FAP-CAR T-cell–treated A549 tumors. Furthermore, versican, a pro-teoglycan implicated in tumor progression that interacts withHA, fibronectin, collagen, as well as cell surface proteins suchas CD44 and integrin-b1 (47, 48), was depleted by 93%(P < 0.01; Fig. 4E). The effects of conditional FAPþ CASCdepletion indicate that these cells are critical for maintenanceof desmoplasia.

FAP-CAR T cells inhibit the growth of nonimmunogenic buthighly desmoplastic PDAs through immune-independentmechanisms

Havingdetermined that depletionof FAPþCASCs could inhibitstromagenesis, angiogenesis, and resolve desmoplasia, we soughtto determine if these findings would extend to PDA, one of the

Figure 3.FAP-CAR T cells restrict tumor growththrough decreasing stromagenesisand tumor angiogenesis indesmoplastic human lung cancerxenografts. Established A549 tumor-bearing mice were either injectedintravenously with FAP-CAR humanT cells or left untreated. Tumors wereharvested 8 days postadoptive T-celltransfer. Tumor sections were stainedwith antibodies against FAP (A) anda-SMA (B) to examine the degree ofstromagenesis. Tumor tissues werestained with antibodies against CD31(C) andNG2 (D) to reveal endotheliumand pericytes, respectively. � ,statistical significance betweenuntreated and FAP-CAR T-cell–treated samples (n ¼ 3), P < 0.01.Scale, 100 mm.

Lo et al.





most desmoplastic tumor types. Furthermore, because FAP-CAR Tcells were able to restrict the growth of human xenografts inimmune-incompetentmice, we also sought to determinewhetherimmune-independentmechanismsmight also play a role in PDA,one of the least immunogenic tumor types described. We initiallyemployed the 4662 tumor cell line that we derived from a tumorthat arose spontaneously in a KPC mouse. 4662 tumors werenonimmunogenic as the growth of this tumor was comparablewhen implanted subcutaneously into syngeneic C57BL/6 versusNSG mice (Supplementary Fig. S5A). Furthermore, antibody-mediated depletion of either CD4þ or CD8þ T cells did notchange tumor growth relative to isotype-treated controls (datanot shown). C57BL/6 or NSG mice bearing established 4662tumors were injected with two doses of FAP-CAR or MigR1 T cells(one week apart). Seven days following the administration of thesecond dose of T cells, the growth of 4662 PDA was reduced by36% (P < 0.05) in immune-competent C57BL/6 mice thatreceived FAP-CAR T cells relative to the tumors frommice treatedwith MigR1 T cells (Fig. 5A). The number of CD11bþLy6Gþ cellswas decreased at this time point in tumors from FAP-CAR T-cell–treated C57BL/6 mice (Supplementary Fig. S5B) and we alsoobserved only a verymodest increase (0.42%) in the total number

of GFP� CD8þ host T cells. Flow cytometric analysis also dem-onstrated infiltration of the adoptively transferred GFPþ FAP-CART cells into 4662 tumors (Supplementary Fig. S5C). Importantly,FAP-CAR T cells had a similar impact on the growth of 4662tumors in immune-incompetent NSG mice, indicating that FAP-CAR T cells inhibited 4662 tumor growth via immune-indepen-dent mechanisms (Fig. 5A).

We next extended these studies to the autochthonous KPCmouse model of PDA. Tumor-bearing KPC mice were treatedwith FAP-CAR or control T cells and tumor growth was mea-sured by ultrasound over a 2-week period. Treatment with FAP-CAR T cells significantly inhibited tumor growth in KPC micecompared with significant tumor progression in the controls(Fig. 5B).

We also assessed the impact of treatment with FAP-CAR T cellson the desmoplastic response and angiogenesis in transplantedand autochthonous PDAs. FAP-CAR T cell treatment alteredstromal architecture, reduced proliferation, and increased apo-ptosis in 4662 and autochthonous PDAs (Fig. 5C and D andSupplementary Fig. S6). FAP-CAR T-cell treatment decreasedFAPþ stromal cells by 62% (P < 0.01) and 91% (P < 0.01) in4662 tumors and autochthonous PDAs, respectively (Fig. 6A).

Figure 4.Depletion of tumor stroma by FAP-CAR T cells in desmoplastic humanlung cancer xenografts. EstablishedA549 tumor-bearing mice weretreated with FAP-CAR human T cells.Tumors were harvested 8 dayspostadoptive T-cell transfer toevaluate the extent of desmoplasia.A, Masson's trichrome staining wasperformed to determine collagenaccumulation. B, IHC was performedto reveal fibronectin content. C, Alcianblue staining was performed to showtotal GAGs. Tumor tissues werestained with HABP (D) or anti-versican antibody (E). � and #,statistical significance betweenuntreated and FAP-CAR T-cell–treated samples (n ¼ 3), P < 0.01 andP < 0.05, respectively. Scale, 100 mm.






Interestingly, FAP and SMA defined distinct, yet overlapping,subsets of stromal cells in both mouse and human pancreatictumors (Supplementary Fig. S7). Similar to the results describedabove, we again observed 70% depletion of SMAþ cells in bothmodels of PDA (Fig. 6B). Furthermore, FAP-CAR T-cell treatmentresulted in a 50% reduction in tumor endothelial cells (Fig. 6C)and 70%decrease in pericytes (Fig. 6D) in bothmodels. Collagencontent in murine PDAs was decreased by 70% to 80% (Fig. 7A)following treatment with FAP-CAR T cells, whereas HA andversican were reduced by 34% to 52% (Fig. 7B) and 61% to74% (Fig. 7C), respectively.

To determine whether FAP-CAR T-cell–mediated disruption oftumor stroma could facilitate drug delivery to inhibit tumorprogression, we treated mice bearing 4662 tumors with FAP-CART cells along with gemcitabine. The combination therapy hadadditive anti-tumor activity compared with the impact of treat-ment with either alone (Supplementary Fig. S8).

Discussion

There is growing interest in developing therapeutic strate-gies that target nonmalignant, tumor-promoting CASCs. Oneemerging candidate is FAP, which is expressed by CAFs, MSCs,and a subset of M2 macrophages, but not by quiescentfibroblasts. Clinicopathological studies indicate that accumu-lation of FAPþ CASCs is associated with tumor progression,metastasis, and predicts poorer outcome in many types ofhuman malignancies, including PDA and colon cancer(49, 50). In contrast, accumulation of SMAþ myofibroblastscorrelates with better prognosis and depletion of these cellsaccelerates tumor progression in murine models of PDA(34). Taken together, these data indicate that deleting FAPþ

CASCs and SMAþ myofibroblasts can have opposing effects,highlighting the phenotypic and functional heterogeneity oftumor stromal cells.

Figure 5.Immune-independent antitumormechanisms play a key role in FAP-CAR T-cell–induced growth inhibitionin murine PDA. A, established 4662tumor-bearing C57BL/6 and NSGmice were treated with FAP-CAR orMigR1 mouse T cells (n¼ 5 per group).A second dose was given 6 days later.� and # denote statistical significancebetween MigR1 and FAP-CAR–treatedsamples in B6 and NSG mice,respectively (P < 0.05). B, tumor-bearing KPC mice received FAP-CARor MigR1 T cells intravenously andtumor progression was followed byultrasound (n ¼ 3 per group). � ,statistical significance between MigR1and FAP-CAR–treated samples, P <0.01. The black arrow indicates thetime of T-cell transfer. 4662 PDAs (C)and autochthonous PDA (D) sectionswere stained with antibodies againstKi-67 and cleaved caspase-3. � and #,statistical significance between MigR1and FAP-CAR T-cell–treated samples,P < 0.01 and P < 0.05, respectively.Scale, 100 mm. H&E, hematoxylinand eosin.

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Several studies showed that depletion of FAPþ CASCsrestricted tumor growth by enhancing antitumor immunity(16, 35, 36, 44). This likely occurs through multiple mechan-isms, including loss of immunosuppressive effects of stromalcells and/or enhancement of tumor access, recruitment, sur-vival, or retention of immune cells. These mechanisms bodewell for combining stromal-targeted CAR T-cell therapy withcancer vaccines or immunological checkpoint antagonists suchas anti-CTLA-4, anti-PD-1, or anti-PD-L1 (16, 35, 36, 44).Here, we explored the possibility that FAPþ CASCs may alsoexert tumor-promoting functions via immune-independentmechanisms by depleting FAPþ CASCs using FAP-CAR T cellsin mouse tumors and human xenografts that exhibit vary-ing degrees of desmoplasia and immunogenicity, and in set-tings in which a potential role for adaptive immunity waseliminated.

Using tumors with varying amounts of desmoplasia, wefound that the extent of matrix accumulation correlated withthe preponderance of FAPþ cells, consistent with previous re-ports that stromal cells are an important source of ECM (1–4).Moreover, our results clearly establish that maintenance oftumor-associated desmoplastic stroma depends on the persis-tent presence of FAPþ cells, because conditional depletion ofthese cells markedly depleted intratumoral ECM. The effects

were transient, however, as stroma returned after the disappear-ance of FAP-CAR T cells (data not shown). Taken together with arecent study of the impact of SMAþ myofibroblast depletion(34), the data presented herein indicate that although bothFAPþ and SMAþ stromal cells regulate intratumoral collagencontent, only FAPþ cells are critical for the maintenance ofintratumoral HA, suggesting that either FAPþ cells are the majorsource of HA or that FAPþ cells are required to drive SMAþ

myofibroblast deposition of HA.The disruption of ECM components known to directly support

tumor cell survival and growth likely underlies FAP-CAR T-cell–mediated growth restrictions of human xenografts (A549) and ofpoorly immunogenic 4662 tumors in immune-incompetent andsyngeneic immune-competent hosts. These effects may be direct,through disruption of stromal cell- and matrix-dependent signal-ing in tumor cells, and/or indirect through inhibition of angio-genesis. These mechanisms, however, are not always sufficient toimpact tumor growth. The growth inhibition of immunogenicAE17.OVA tumors by FAP-CAR T cells was lost in immuneincompetent hosts, indicating a requirement of adaptive immu-nity (44). However, AE17.OVA tumors grew much more rapidlyin NSG mice (44). Such an increase in growth kinetics mightovercome any potential immune-independent tumor inhibitoryeffects elicited by FAP-CAR T cells in a tumor that exhibits

Figure 6.FAP-CAR T cells impact stromagenesis and angiogenesis in murine PDA. Established 4662 tumor-bearing mice (n ¼ 6 per group) and autochthonousPDA-bearing KPC mice (n ¼ 3 per group) were treated with two doses of FAP-CAR mouse T cells. IHC was performed using tumor tissues harvested frommice 3 days after the second dose of T-cell transfer. Tumor sections were stained with antibody against FAP (A), SMA (B), CD31 (C), and NG2 (D). � and#, statistical significance between MigR1 and FAP-CAR T-cell–treated samples, P < 0.01 and P < 0.05, respectively. Scale, 100 mm.






minimal stromagenesis. Moreover, FAP-CAR T-cell–mediatedmatrix modification may have augmented antitumor immunityby influencing immune cell recruitment and/ormotility, asmatrixarchitecture has been shown to regulate intratumoral T-cell local-ization and migration (24). We found that depletion of FAPþ

CASCs modestly increased CD8þ T-cell infiltration even intononimmunogenic highly desmoplastic 4662PDAs, but their rolesin inhibiting tumor growth in this model remain to be deter-mined. In addition, we observed neither an increase in Treginfiltration, nor importantly, increased incidence of lung metas-tasis in the tumor-bearingmice treatedwith FAP-CAR T cells (datanot shown).

In addition to immunomodulatory effects, stromagenesis hasdirect effects on tumor cells and indirectly controls tumor growththrough regulation of angiogenesis (7, 11).We found that CASCs,including FAPþ cells and SMAþmyofibroblasts, were significantlydecreased in A549, 4662, and autochthonous PDA tumors post-FAP-CAR T-cell transfer. In addition, tumor angiogenesis wasprofoundly diminished. Taken together, our data demonstratethat FAP-CAR T cells inhibit tumor stromagenesis and reducevascular density and/or integrity in highly desmoplastic tumors.Based on the small degree of overlap between FAPþ and SMAþ

cells in A549, 4662 tumors and autochthonous PDA, it is unlikelythat FAP-CAR T cells directly targeted myofibroblasts. The reduc-tion in myofibroblasts that results from depletion of FAPþ cellssuggests that either SMAþFAP� cells are derived from MSCs thatexpress FAPþ at an earlier stage in their differentiation and/or thatFAPþ cells are required to induce the recruitment and/or differ-entiation of SMAþ myofibroblasts (1, 27, 33). It will therefore beinteresting to further define the lineage and functional relation-

ships among FAPþ stromal cells, myofibroblasts, and the pancre-atic stellate cells thought to be critical to pancreatic fibrosis.

An unexpected observationwas themarked impact of FAPþ celldepletion on tumor cell morphology. In AE17.OVA tumors,depletion of FAPþ cells resulted in signs of nuclear condensation.However, we did not observe immune-mediated hypoxia-asso-ciated necrosis as previously reported (36). Instead, tumors thatreceived FAP-CAR T-cell treatment exhibited a reduced prolifer-ative index and an increased apoptotic index compared witheither MigR1 T-cell–treated or untreated tumors. In addition,FAP-CAR T-cell treatment disrupted tumor ductal-like structure,indicating that extrinsic cues provided by stroma are critical to thespatial orientation of tumor cells.

Recent studies raised the concern that ablation of FAPþ stromalcells could induce bone marrow hypocellularity and cachexia inmice (37, 43). Despite careful examination, we again did notobserve bonemarrow destruction or changes in total body weightusing the indicated treatment regimens in the tumor modelsinvestigated. The difference in toxicities may be due to thedifference in epitope specificity of the anti-FAP antibodies usedto generate CARs in different studies, the fact that our FAP-CAR Tcells only transiently deplete FAPþ cells and/or the fact that theFAP-CAR T cells we generated preferentially deplete cells expres-sing high levels of FAP (including tumor-associated stromal cells)but not cells expressing low levels of FAP (e.g., basal levels of FAPon normal cells). Thus, as we previously proposed (44), theremight be a therapeutic windowusing appropriate CAR constructs.Results from other research groups (38, 39, 41), including twoindependently generated FAP-CAR constructs (40, 42) also sup-port this concept.

Figure 7.FAP-CAR T cells resolve desmoplasia in murine PDA. Established 4662 tumor-bearing mice (n ¼ 6 per group) and autochthonous PDA-bearing KPCmice (n ¼ 3 per group) were treated with two doses of FAP-CAR or MigR1 mouse T cells. Tumor tissues were harvested 3 days after the second dose ofT cell transfer to evaluate the extent of desmoplasia. A, collagen was evaluated by Masson's trichrome staining. B and C, HA was evaluated by HABP(B) and versican (C) determined by immunohistochemistry staining. � and #, statistical significance between MigR1 and FAP-CAR T-cell–treated samples,P < 0.01 and P < 0.05, respectively. Scale, 100 mm.

Lo et al.





Given potential toxicity, it is important to note that even atemporary disruption of tumor stroma may be sufficient fortherapeutic benefit. For instance, transient depletion of stromalHA was shown to facilitate delivery of gemcitabine to tumors,thereby augmenting efficacy of chemotherapy inpancreatic cancer(18, 20). Previous work employing anti-FAP vaccination resultedin decreased levels of collagen 1 and increased intratumoraluptake of chemotherapeutics (39). Furthermore, we and othersshowed that depletion of FAPþ CASCs could disrupt immuno-suppression to enhance therapeutic efficacy of cancer vaccines(36, 44) or checkpoint antibodies (16). We postulate that thedesmoplastic response mediated by FAPþ CASCs could thuscontribute to therapeutic resistance through themultiplemechan-isms discussed here.We are currently testing if depletion of tumorstroma by FAP-CAR T cells could augment the efficacy of multipleother therapeutic modalities. Our data to date indicate that thecombination of stromal cell depletion with gemcitabine treat-ment provides at least additive anti-tumor effects in 4662 tumorsand we are exploring the mechanisms involved.

In summary, the results presented herein provide proof-of-concept that, in addition to suppressing adaptive anti-tumorimmunity, FAPþ CASCs orchestrate tumor-promoting effects byenhancing tumor angiogenesis and desmoplasia. The balance ofthese various tumor-promoting effects of FAPþ cells is determinedby multiple parameters: the immunogenicity of the tumor, theprevalence of FAPþCASCs and extent of desmoplasia in the tumormicroenvironment. Furthermore, depletion of FAPþ CASCs inhi-bits tumor growth through both adaptive immune-dependentand -independent mechanisms. Moreover, cross-talk betweenthese mechanisms may also contribute to reduced tumor growth.Therefore, developing combinatorial strategies that incorporateFAPþ CASC depletion for cancer treatment may offer therapeuticadvantages.

Disclosure of Potential Conflicts of InterestR.H. Vonderheide reports receiving commercial research grants from Roche

and Pfizer. C.H. June, S.M. Albelda, and E. Pur�e report receiving a commercialresearch grant fromNovartis. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: A. Lo, L.-C.S. Wang, J. Scholler, S.M. Albelda, E. Pur�eDevelopment of methodology: L.-C.S. Wang, J. Scholler, J. Monslow,R.H. Vonderheide, E. Pur�eAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Lo, L.-C.S. Wang, J. Monslow, D. Avery, K. Newick,S. O'Brien, R.A. Evans, D.J. Bajor, C. Clendenin, A.C. Durham, C.H. June,S.M. Albelda, E. Pur�eAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Lo, L.-C.S. Wang, D. Avery, R.H. Vonderheide,S.M. Albelda, E. Pur�eWriting, review, and/or revision of the manuscript: A. Lo, L.-C.S. Wang,D. Avery, E.L. Buza, R.H. Vonderheide, C.H. June, S.M. Albelda, E. Pur�eAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Pur�eStudy supervision: E. Pur�eOther (histopathologic interpretation): E.L. Buza, A.C. Durham

AcknowledgmentsThe authors thankRajrupa S.Majumdar andVeenaKapoor for their technical

support. The authors also thank Dr. Ben Stanger for helpful discussions andMouse Hospital at the University of Pennsylvania for providing KPC mice. Theauthors also thank Boehringer Ingleheim for providing FAPLacZ knock-in miceand histology facility at the Ryan Hospital of the School of Veterinary Medicineat the University of Pennsylvania.

Grant SupportThese studies were supported by NCI (National Cancer Institute) grants P01

CA 66726-07 (S.M. Albelda and C.H. June), R01 CA169123 (R.H. Vonder-heide), R21CA169741, R01CA141144, andR01CA172921 ( S.M. Albelda andE. Pur�e), and a "Clinic and Laboratory Integration Program" Grant from theCancer Research Institute (E. Pur�e and S.M. Albelda) and from NovartisPharmaceuticals Corporation, and the Lung Cancer and Pancreatic CancerTranslational Centers of Excellence at the Abramson Cancer Center. A. Lo wassponsored by a START (Student Training and Research in Tumor Immunology)fellowship from the Cancer Research Institute. L.-C.S. Wang was supported byPittsburgh 5K Cure Sarcoma Award.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 17, 2014; revised May 6, 2015; accepted May 7, 2015;published OnlineFirst May 15, 2015.

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Lo et al.




2015;75:2800-2810. Published OnlineFirst May 15, 2015.Cancer Res Albert Lo, Liang-Chuan S. Wang, John Scholler, et al. FAP-Expressing Stromal CellsTumor-Promoting Desmoplasia Is Disrupted by Depleting

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