CD8 T Cells Regulate Bone Tumor Burden Independent of Osteoclast

Published OnlineFirst May 20, 2011; DOI: 10.1158/0008-5472.CAN-10-3922

Cancer Microenvironment and Immunology Research

CD8þ T Cells Regulate Bone Tumor Burden Independent of Osteoclast Resorption

Kaihua Zhang1, Seokho Kim1, Viviana Cremasco1, Angela C. Hirbe2, Deborah V. Novack3, Katherine Weilbaecher2, and Roberta Faccio1

Abstract Blockade of osteoclast (OC) activity efficiently decreases tumor burden as well as associated bone erosion in immune-compromised animals bearing human osteolytic cancers. In this study, we showed that modulation of antitumor T-cell responses alters tumor growth in bone, regardless of OC status, by using genetic and / pharmacologic models. PLCg2 mice, with dysfunctional OCs and impaired dendritic cell (DC)–mediated / T-cell activation, had increased bone tumor burden despite protection from bone loss. In contrast, Lyn mice, with more numerous OCs and a hyperactive myeloid population leading to increased T-cell responses, had reduced tumor growth in bone despite enhanced osteolysis. The unexpected tumor/bone phenotype observed in PLCg2 / and Lyn / mice was transplantable, suggesting the involvement of an immune component. Consistent with this hypothesis, T-cell activation diminished skeletal metastasis whereas T-cell depletion enhanced it, even in the presence of zoledronic acid, a potent antiresorptive agent. Importantly, injection of þ / þ / antigen-specific wild-type cytotoxic CD8 T cells in PLCg2 mice or CD8 T-cell depletion in Lyn mice þ normalized tumor growth in bone. Our findings show the important contribution of CD8 T cells in the regulation of bone metastases regardless of OC status, thus including T cells as critical regulators of tumor growth in bone. Cancer Res; 71(14); 4799–808. 2011 AACR.

Introduction ment with zoledronic acid (ZOL), an N-bisphosphonate com- pound highly effective in inducing OC apoptosis and Bone metastases represent a serious complication of many suppressing bone resorption, protects mice injected with cancers, including breast, prostate, and lung tumors, as well as human breast cancer cells from developing tumor growth in multiple myeloma. In the bone marrow (BM), there is a bone (3). Based on these findings, the OC is now the principal mutually positive interaction between osteoclasts (OC) and therapeutic target for bone metastases (4). However, not all cancer cells that is known as the vicious cycle (1). Growth patients with bone metastases respond well to antiresorptive factors secreted by the tumor cells, such as receptor activator therapy, and one third develops further skeletal-related events of NF-kB–ligand (RANKL), stimulate the bone resorptive within 2 years of initiating these therapies (5). Thus, the results activity of OCs. In turn, bone-stored factors including TGF- from clinical studies suggest that other bone marrow–residing b are released and enhance tumor growth (1). Experimental cells, in addition to OCs, could be regulating tumor growth in evidence suggests that OCs play a central role in modulating bone. the tumor/bone vicious cycle. Antagonism of RANKL, by using The bone microenvironment is a reservoir of several recombinant osteoprotegerin (Fc-OPG) or soluble anti- immune cell types. Memory T cells have been found in the RANKL antibody (Ab), reduces OC number and significantly bone marrow of patients with breast cancer, implicating them decreases tumor burden in murine models of breast cancer in cancer immune surveillance (6). Interestingly, some of the bone metastasis and multiple myeloma (2). Similarly, treat- therapies aimed at disrupting the mutual interaction between cancer cells and OCs also have immunomodulatory effects. For example, TGF-b, which is released into the bone marrow Authors' Affiliations: Departments of 1Orthopedics, 2Molecular Oncol- microenvironment by the resorptive OC, inhibits T-cell pro- 3 ogy, and Medicine, Washington University School of Medicine, St. Louis, liferation, natural killer (NK) cell function, and antigen pre- Missouri sentation (7). Thus, blockade of TGF-b at sites of metastases Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). may locally activate T-cell function, initiating an antitumor immune response. ZOL, in addition to its antiresorptive effect, K. Zhang and S. Kim contributed equally to the manuscript. can activate cytotoxic g/d-T cells (8) and inhibit certain Corresponding Author: Roberta Faccio, Department of Orthopedics, – Washington University School of Medicine, Campus Box 8233, 660 S populations of myeloid-derived cells with T-cell suppressor Euclid, St. Louis, MO 63110. Phone: 314-747-4602; Fax: 314-362-0334; abilities. Unfortunately, to date, the contribution of T cells in E-mail: [email protected] modulating the tumor/bone vicious cycle has not been eval- doi: 10.1158/0008-5472.CAN-10-3922 uated because most models of bone metastases use human 2011 American Association for Cancer Research. breast cancer cells injected into immunocompromised mice.

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The present study aimed to examine the relative contribu- Firefly-conjugated B16 cells (B16-FL) were injected intrati- tion of immune cells and OCs in the tumor/bone vicious cycle bially (IT), intracardiac (LV), or subcutaneously (s.c.) in 6- by using a syngenic mouse model. We turned to the B16 week-old, female mice (ref. 21; Supplementary Data). For þ þ melanoma model of bone metastases because these cells: (i) CD4 or CD8 T-cell depletion we injected 100 mg/100 mL grow in C57BL/6 immunocompetent mice, and (ii) metasta- YTS191.1.2 or YTS169.4.2.1 mAb, respectively, starting 1 day size to bone following intracardiac injection. B16 is a relatively before tumor inoculation and continuing every other day for poorly immunogenic cell line, although it can induce a mod- the duration of the experiment. ZOL (0.75 mg/mouse) was est, but specific, T-cell response (9). Thus, by using B16 cells administered s.c. 10 and 4 days before tumor inoculation, and we can take advantage of genetically manipulated mice with mice were sacrificed 2 weeks later. This dosing of ZOL was specific immune phenotypes. Phospholipase C gamma (PLCg) designed to produce drug levels similar to those achieved with 2 is an enzyme converting phosphatidylinositol 4,5-bispho- the clinical dosing regimen of 4 mg Zometa for the treatment sphate (PIP2) into diacylglycerol (DAG) and inositol tripho- of bone metastases (22). sphate (IP3) leading to activation of protein kinase C (PKC) / and calcium pathways. PLCg2 mice have broadly compro- Bone histomorphometry mised immune responses due to impaired B-cell development, Mouse tibias were decalcified and processed for bone NK-cell cytotoxic activity, and dendritic cell (DC)-mediated histomorphometry as described previously (21). Because / / antigen presentation leading to defective T-cell activation PLCg2 and Lyn mice have different basal bone mass / (10–13). Furthermore, PLCg2 mice are osteopetrotic due compared with wild-type (WT) mice, we calculated bone loss to reduced OC number and functionality (14, 15). Lyn is a Src as percentage of bone volume (BV)/total bone volume (TV) family member that is mainly involved in down-modulation of relative to their tumor-free controls (arbitrarily set at 100%) several intracellular pathways, including PLCg2 activation using Bioquant Osteo. / (16). Lyn mice have increased B-cell–mediated immune responses, expanded macrophages, mast cells, and DCs (17, Bone marrow transplantation 18). Due to a hyperactive myeloid population, T-cell responses Female, 5-week-old C57BL/6 mice were lethally irradiated / are also enhanced, and Lyn mice develop autoimmunity using a 137Cs source with 900 rads to generate recipient / with age (19). Furthermore, Lyn mice have decreased bone mice. Bone marrow was harvested from 6-week-old, female / / mass and more numerous OCs due to enhanced RANKL PLCg2 and Lyn mice or WT littermates, suspended in signaling and PLCg phosphorylation (16, 20). PBS, and 200 mL containing a concentration of 106 cells was Because PLCg2 deficiency impacts OC formation and injected into the lateral tail vein of recipient mice to generate / / / function, we expected that PLCg2 mice would have PLCg2 and Lyn radiation chimeras and WT controls. decreased bone tumor burden following B16 tumor inoculation. Furthermore, we would have anticipated increased DC generation / tumor growth in bone in Lyn mice on the basis of their DCs were generated from bone marrow progenitor cells hyperactive OC phenotype. On the contrary, we found that from 5- to 8-week-old C57BL/6 mice, as previously described / PLCg2 micearemoresusceptibletotumorgrowthin (23; Supplementary Data). DCs were matured with 1 mg/mL / bone, despite their OC defect, whereas Lyn mice display lipopolysaccharide (LPS) overnight, and upregulation of significant inhibition of tumor growth in bone, in the mature DC markers was confirmed by fluorescence-activated face of more numerous OCs. Despite the lack of PLCg2 cell sorting (FACS). and Lyn expression in T cells, aberrant myeloid-mediated T-cell activation is responsible for these unexpected find- T-cell adoptive transfer þ þ ings. Our data show that CD8 T cells modulate tumor CD8 T cell were magnetically isolated from WT mice growth in bone regardless of genetic or pharmacologic OC vaccinated with GP10025–33 (10 mg/mL; Bio-Synthesis Inc.) þ inhibition, thus expanding the current tumor/bone vicious pulsed DCs by using CD8 T Cell Isolation Kit (Miltenyi þ cycle model to include an immune component in addition Biotec). Reactivity of GP100-specific CD8 T cells was con- to the OC. firmed in vitro by IFN-g release following peptide stimulation. þ A sample of 1 106 CD8 T cells plus 200 mL of interleukin Materials and Methods (IL)-2 (2,000 U/mL; Chemicon) were adoptively transferred / into tumor-bearing (IT-injected) PLCg2 mice via tail vein. Tumor cells and animal models of bone metastases Subsequently, GP10025–33 peptide-pulsed (4 hours), LPS- B16 mouse melanoma cells were obtained from Dr. David matured DCs were s.c. administered on the day of the adoptive þ Fisher (Harvard Medical School). These cells have been char- transfer to further activate GP100-specific CD8 T cells. / acterized by expression arrays and Western blot analysis for Tumor-bearing WT and PLCg2 mice receiving IL-2 alone melanocyte markers such as melanin, tyrosinase, c-kit, and were used as controls. Tumor growth was monitored on days microphtalmia transcription factor. Pigmented tumor forma- 8, 10, and 12 by bioluminescence imaging (BLI). tion in mice further confirmed that B16 are melanoma cells. þ This assay is routinely used in the laboratory for their char- Detection of GP10025–33-specific CD8 T cell acterization and it was conducted within the last few weeks of Spleens were isolated from B16-FL s.c. injected mice. Sple- the experiment. nocytes were cultured with 10 mg/mL GP10025–33 peptide, in

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Contribution of CD8þ T Cell in Bone Tumor Burden

the presence of Golgistop (Becton Dickinson), to inhibit populations. Results were considered significant at P < 0.05, cytokine secretion. After 8 hours, samples were stained with and are indicated with an asterisk (*). a-CD8-fluorescein isothiocyanate (FITC) Ab (Becton Dickin- son), fixed, permeabilized using Cytofix/Cytoperm Kit (Becton Results Dickinson), co-stained with R-phycoerythrin (PE)-conjugated a-IFN-g Ab, and analyzed by FACS. PLCg2 / mice have increased bone tumor burden despite defective OC function Cytometric bead array Deletion of PLCg2 in mice results in an osteopetrotic A sample of 5 104 splenocytes from B16-FL s.c.-injected phenotype due to OC defects (14, 15). Based on the tumor/ mice was plated in a 96-well/plate with 200 mL of RPMI-1640 bone vicious cycle model in which OC activity is required to / and 10% mouse serum. Cells were restimulated with irradiated sustain tumor growth in bone, we predicted that PLCg2 B16-FL (2,500 rads) for 3 days. Supernatants were harvested mice would be protected from bone metastases. To test this and IFN-g production was assessed by FACS analysis using the hypothesis, we injected luciferase-labeled B16 cells (B16-FL) TH1/TH2 Assay Kit (Becton Dickinson) according to the IT, to test tumor growth in bone, or into the left cardiac manufacturer's instructions. ventricle (LV), to test tumor tropism to bone, of 6-week-old / PLCg2 and WT littermates. Presence of bone tumors was Statistical analysis measured by BLI 8 and 10 days after tumor inoculation, and by For each in vivo experiment, 4 to 6 mice per group were histologic examination on day 12. Unexpectedly, despite the / used. Experiments were done in triplicate and analyzed using OC defect, PLCg2 mice showed significantly increased Student's t-test. In calculating 2-tailed significance levels for tumor burden in bone (Fig. 1A). Histologic analysis confirmed / equality of means, equal variances were assumed for the 2 the increase in tumor area in PLCg2 bones (Fig. 1B and C).

A LV IT B

200 –/– WT PLCγ2 ** 300 WT 160 WT * PLCγ2–/– PLCγ2–/– T T 200 120 80 photons/s)

4 100 Total flux Total 40 T ( × 10 0 0 810 8 10 (days) C LV IT D LV IT 0.6 * 6 * 0.6 4 0.4 4 0.4

OCs/ 2 0.2 0.2 2 * * bone perimeter

Tumor areal/total area Tumor 0 –/– 0 –/– 0 0 –/– WT PLCγ2 WT PLCγ2 WT PLCγ2–/– WT PLCγ2 EFLV IT

3 * * 80 80 2

40 40 CTX * 1 tumor CTR (fold induction) (fold % BV/TV vs. no % BV/TV vs. 0 0 0 –/– –/– WT PLCγ2–/– WT PLCγ2 WT PLCγ2

Figure 1. PLCg2 / mice display increased tumor burden in bone, but not tumor-associated bone loss. B16-FL cells were injected LV or IT into 6-week-old WT and PLCg2 / littermates. A, BLI at days 8 and 10 showed higher tumor burden in PLCg2 / mice (LV: **, P < 0.001 vs. WT mice; IT: *, P < 0.05 vs. WT mice; n ¼ 4). B, tartrate-resistant acid phosphatase (TRAP)–stained sections from WT and PLCg2 / tibias showed OCs (red cells in 2.5 magnification insert) and tumor cells (T). C, histologic analysis of tumor area/total bone area showed a significant increase in tumor growth in PLCg2/ mice (*, P < 0.05 vs. WT) despite decreased OC number/bone perimeter (D;*,P < 0.05 vs. WT). E, bone loss was calculated as the percentage of BV/TV relative to tumor-free controls (CTR). Although WT mice underwent 50% bone loss after tumor injection, only less than 20% bone loss occurred in PLCg2 / mice (*, P < 0.05 vs. WT). F, increased bone loss in WT mice was further confirmed by higher CTX levels. Data are expressed as fold induction from baseline, arbitrarily set as 1, and significant lower CTX levels in PLCg2 / mice are indicated (*, P < 0.05 vs. WT; n ¼ 5forWT and n ¼ 4forPLCg2 / ).

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/ These results were also replicated using non-labeled parental increased bone tumor burden in PLCg2 mice was mediated B16 cells (data not shown), indicating that the presence of the by cells of the hematopoietic compartment. We transplanted / luciferase was not responsible for the altered tumor growth. bone marrow cells from WT or PLCg2 donors into lethally Interestingly, consistent with impaired basal osteoclasto- irradiated 5-week-old C57BL/6 WT mice. Three weeks after / genesis, tumor-bearing PLCg2 animals continued to dis- transplantation, B16-FL cells were inoculated LV. Similar to / play a 3-fold decrease in OC number compared with equally PLCg2 mice, tumor growth in bone was increased in WT / / treated WT littermates (Fig. 1D). Despite the increase in tumor mice carrying PLCg2 marrow cells (PLCg2 > WT), as / burden, PLCg2 mice were still protected from tumor- determined by BLI (Fig. 2A) and histologic analysis (Fig. 2B / associated bone destruction as determined by more than and C). PLCg2 > WT-transplanted mice were protected 80% of remaining trabecular BV/TV relative to their tumor- from tumor-induced bone loss, and the number of OCs per free controls. In contrast, tumor-bearing WT mice displayed bone perimeter was approximately 4-fold less compared with approximately 50% reduction in BV/TV (Fig. 1E). Decreased WT > WT transplants (Fig. 2D and E). Based on the dysfunc- / / tumor-associated bone erosion in PLCg2 mice was further tional PLCg2 OC phenotype, these findings indicate that confirmed by low serum levels of Collagen type I fragment cells of hematopoietic origin, other than OCs, contributed to / (CTX), a marker of in vivo OC activity (Fig. 1F). Thus, our data enhance tumor growth in the bone of PLCg2 mice. show that OC deficiency is not sufficient to prevent tumor / growth in bone of PLCg2 mice. Lyn / mice are protected from bone tumor growth despite increased OC responsiveness Increased bone tumor burden in PLCg2 / mice is To determine whether an aberrant immune condition transplantable might contribute to regulation of tumor growth in bone / / Because PLCg2 mice have broadly compromised mye- regardless of the OC status, we turned to Lyn mice, loid and NK cell functions (10–13), we explored whether the characterized by enhanced myeloid cell and OC functionality

A B WT>WT γ –/– 500 ** PLC 2 >WT WT>WT 400 –/– PLCγ2 >WT T 300 T

photons/s) 200 4 Total flux Total T Figure 2. Increased bone tumor

( × 10 100 burden in PLCg2 / mice is 0 transplantable. Three weeks after 8 10 (days) transplantation, B16-FL were C D injected LV into WT mice 0.7 3 transplanted with WT (WT > WT)or * with PLCg2 / (PLCg2 / > WT) 0.6 bone marrow cells. A and B, BLI 0.5 2 and TRAP-stained long bone sections showed increased tumor 0.4 growth in bone of PLCg2 / > WT 0.3 (**, P 0.01 vs. WT WT mice; n ¼ 1 * < > 0.2 6; OCs are visualized in insert 2.5 magnification). Histomorphometry

0.1 OCs/bone perimeter / Tumor area/total area Tumor in PLCg2 > WT transplanted 0.0 0 animals revealed (C) increased WT>WT γ –/– –/– E PLC 2 >WT WT>WT PLCγ2 >WT tumor area/total bone area (*, P < WT WT 100 * 0.05 vs. > mice), (D) reduced OC number (*, P < 0.05 WT WT 80 vs. > mice), and (E) reduced tumor-induced bone loss P WT WT 60 (*, < 0.05 vs. > mice).

%BV/TV vs. no tumor CTR %BV/TV vs. 0 WT>WT PLCγ2–/– > WT

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A B

LV IT –/– 400 600 WT Lyn WT 300 WT Lyn–/– Lyn–/– 400 Lyn/ Figure 3. animals are 200 * * T protected from tumor burden in photons/s) 4

Total flux Total 200 bone but not from bone loss. A 100 and B, BLI and TRAP-stained long ( × 10 bones showed reduced tumor 0 0 growth (T) in bones of Lyn/ mice 8108 10 (days) inoculated LV or IT with B16-FL CDLV IT * cells (*, P < 0.05 vs. WT mice; n ¼ LV IT * 0.8 0.6 6). C, histomorphometry showed decreased tumor area/total bone 6 6 0.6 area in Lyn / mice (*, P < 0.05 vs. 0.4 4 WT mice; n ¼ 6), despite (D) 0.4 4 increased OC numbers (*, P < 0.05 * * 0.2 2 vs. WT mice; n ¼ 6; insert 2.5 0.2 2 magnification) and (E) 50% bone Tumor area/total area Tumor 0 0 0 OCs/bone perimeter 0 loss calculated as percentage WT Lyn–/– WT Lyn–/– WT Lyn–/– WT Lyn–/– BV/TV relative to tumor-free controls (CTR). F, CTX serum E F LV IT levels showed higher OC activity 80 80 3 * in Lyn/ mice compared with WT (*, P < 0.05 vs. WT mice; n ¼ 5 2 for WT and n ¼ 4forLyn/). 40 40 1 tumor CTR %BV/TV vs. no %BV/TV vs.

CTX (fold induction) CTX (fold 0 0 0 –/– –/– WT Lyn–/– WT Lyn WT Lyn

/ / (17–19). In contrast to PLCg2 animals, Lyn mice dis- topoietic origin, other than OCs, contribute to reduced tumor / played significant inhibition of tumor growth in bone follow- growth in bone of Lyn mice. ing either intratibial or intracardiac tumor inoculation (Fig. 3A and B). Analysis of tumor area/total bone area confirmed a 4- Modulation of T-cell activation alters tumor burden in / fold decrease in tumor burden in Lyn mice (Fig. 3C). bone independent of OC activity However, despite the significantly reduced bone tumor bur- T cells are known to be modulators of antitumor immune / den, Lyn mice had increased OC numbers and showed as responses (24). To determine whether T cells might be reg- much tumor-associated bone loss as WT mice (Fig. 3D and E). ulating tumor growth in bone, we injected WT mice with the / Consistent with this finding, Lyn mice had elevated serum a-CTLA4 Ab, which enhances T-cell proliferation and/or CTX levels (Fig. 3F). These data indicated that increased OC function (25), following intracardiac inoculation of B16-FL. responsiveness is not sufficient to enhance tumor growth in a-CTLA4 Ab significantly suppressed tumor growth in bone, / bone of Lyn mice. as shown by BLI and histomorphometric analysis (Fig. 5A and B). Conversely, Nude mice, which lack all T-cell subsets, had Reduction in bone tumor burden in Lyn / is enhanced tumor growth in bone compared with control transplantable animals (Fig. 5C). Histologic analysis confirmed an abundance To determine whether cells of the hematopoietic compart- of tumor cells in the bones of Nude mice compared with their ment were responsible for reduced bone tumor burden in controls (Fig. 5D). To determine which T-cell subset might be / / Lyn mice, we carried out bone marrow transplants, in regulating tumor growth in bone, we turned to MHCI mice, / þ / which WT recipient mice carried WT (WT > WT)orLyn which do not have CD8 T cells, and MHCII mice, which / / þ þ þ (Lyn > WT) marrow cells. We found that Lyn > WT lack CD4 T cells. Absence of either CD4 or CD8 T cells transplanted animals were protected from B16-FL tumor significantly increased B16-FL tumor growth in bone following / growth in bone, as determined by BLI (Fig. 4A) and histologic intracardiac inoculation. MHCI mice had >4–fold increase / / / analysis (Fig. 4B). Similar to Lyn mice, Lyn > WT and MHCII mice had >2.5-fold increase in bone tumor transplants displayed disproportionate bone loss for their burden by day 10 as compared with WT mice (Fig. 5E and F). tumor burden, showing similar OC number and percentage Because the tumor/bone vicious cycle model considers OC of BV/TV as WT > WT transplants despite the 4-fold decrease activity to be central to tumor proliferation in bone, we sought in bone tumor burden (Fig. 4C–E). Due to the hyperactive to determine the relative contribution of OCs and T cells in / Lyn OC phenotype, these data indicate that cells of hema- this process. We tested the efficacy of ZOL antiresorptive

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A B WT>WT Lyn–/–>WT 600 WT>WT Lyn–/–>WT 400 T * photons/s) 4 Total flux Total 200

( × 10 Figure 4. Reduction in bone tumor burden in Lyn/ is transplantable. 0 Three weeks after transplantation, 0 10 (days) B16-FL were injected LV into WT C mice transplanted with WT 0.6 D 6 (WT > WT)orLyn/ (Lyn/ > WT) bone marrow cells. A, BLI and B, histology showed reduced tumor 0.4 4 cells (T) in Lyn / > WT mice (*, P < 0.05 vs. WT > WT mice; * n ¼ 4; insert 2.5 magnification). 0.2 2 C, histomorphometry showed reduction in tumor area/total bone Lyn/ WT

nOC/bone perimeter area in > mice Tumor area/total area Tumor 0 0 (*, P < 0.05 vs. WT > WT mice; –/– –/– WT>WT Lyn >WT WT>WT Lyn >WT n ¼ 4), but (D and E) WT > WT and E Lyn / > WT mice showed similar differences in OC number (nOC) 80 and in the percentage of BV/TV relative to tumor-free mice (CTR).

40 %BV/TV vs. CTR %BV/TV vs. 0 WT>WT Lyn–/–>WT

treatment in providing protection from tumor growth in bone 2 weeks after B16-FL s.c inoculation. We examined IFN-g þ þ in mice depleted of CD4 or CD8 T cells. WT mice were release by activated T cells, restimulated in vitro for 3 days treated with ZOL 10 and 4 days before B16-FL intratibial with irradiated B16-FL cells. This assay showed impaired IFN- þ þ / / inoculation, and a-CD4 or a-CD8 T-cell–depleting Abs g release in PLCg2 and hyper-production in Lyn cul- were administered 1 day before and every 2 days after injec- tures compared with WT cultures (Fig. 6A). Furthermore, tion of cancer cells. Efficiency of T-cell depletion was con- IFN-g release was observed in response to parental B16 cells firmed at the end of the experiment by FACS analysis. Despite (data not shown). To further determine whether the antitu- blockade of tumor-associated bone loss (Supplementary Data mor T-cell response was specific to the B16 cell line, spleno- 1), we found that the antitumor effect of ZOL was significantly cytes isolated from tumor-bearing mice were stimulated for þ þ reduced in CD8 , and to a lesser extent in CD4 , T-cell– 8 hours with tumor antigen GP100, expressed by B16 cells. þ depleted mice compared with WT mice receiving the FACS analysis showed that the percentage of CD8 /IFN-g / antiresorptive treatment (Fig. 5G and H). Thus, T cells modu- producing PLCg2 T cells responding to GP100 was signi- / late tumor growth in bone regardless of the OC status. ficantly less than those in WT mice, whereas in Lyn mice it was 2-fold more than that in WT mice (Fig. 6B), supporting / / T-cell abnormalities are responsible for tumor growth the conclusion that PLCg2 and Lyn mice have altered in bone of PLCg2 / and Lyn / mice T-cell responses to tumor. Despite a lack of PLCg2 and Lyn expression in T cells, To further determine whether T-cell abnormalities were / / myeloid cell abnormalities [impaired DC function in PLCg2 responsible for increased bone tumor burden in PLCg2 / (23), myeloid hyperactivity in Lyn mice (19)] could mice, we adoptively transferred tumor-specific cytotoxic þ / induce aberrant T-cell–mediated antitumor responses. To CD8 T cells into PLCg2 mice. Because B16 melanoma determine the presence of activated T cells in tumor-bearing cells express the antigen GP100, we generated GP100-reactive / / þ mice, we isolated spleens from PLCg2 and Lyn mice CD8 T cells by injecting twice GP10025–33 peptide-pulsed

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A B Day 12 800 0.6 WT + IgG Ab WT + α-CTLA4 Ab 600 0.4 400 photons/s) 4

Total flux Total 0.2 * 200 ( × 10

* area/total area Tumor 0 0 8 10 (days) IgG Ab α-CTLA4 Ab Figure 5. T cells modulate tumor C D growth in bone. A and B, BLI and 0.6 Day 12 * histology of tumor area/total bone 400 CTR area showed reduced tumor Nude growth in WT mice with a-CTLA4 300 * 0.4 Ab (T-cell–activating Ab) compared with isotype IgG (*, P < 200 photons/s)

WT n ¼ 4 0.05 vs. mice; 4). C and D, flux Total 0.2 BLI and histology of Nude mice 100 injected LV with B16-FL showed ( × 10 Tumor area/total area Tumor increased tumor growth in bone 0 0 compared with controls (CTR; 8 10 (days) CTR Nude *, P < 0.05 vs. CTR mice; n ¼ 4). E E F and F, BLI and histology in WT, Day 12 400 0.6 MHCI / , and MHCII / mice WT injected LV with B16-FL showed –/– * MHCII ** enhanced tumor growth in bone in MHCI–/– * the absence of specific T-cell 0.4 P WT subsets (**, < 0.01 vs. mice; 200 *, P < 0.05 vs. WT mice; n ¼ 4). * photons/s) 4 G and H, BLI and tumor area/total flux Total 0.2 þ area showed that a-CD4 and

þ ( × 10 a-CD8 Abs reduced the Tumor area/total area Tumor antitumor effect of ZOL compared 0 0 to WT treated with ZOL 8 10 (days) WT MHCI–/– MHCII–/– [**, P < 0.02 immunoglobulin G (IgG) vs. CTR and ##, P < 0.02 IgG G 10 d H vs. a-CD8]. 600 12 d ** ## Day 12

400 * * 0.4 * 0.3 photons/s) 4 Total flux Total 200 0.2

( × 10 0.1 0 0 CTR IgG α-CD4 α-CD8 area/total area Tumor CTR IgG α-CD4 α-CD8 + ZOL + ZOL

þ / mature DCs into WT mice. Two weeks later, CD8 T cells were PLCg2 mice compared with null mice receiving IL-2 alone þ isolated from spleens and their functionality was confirmed in (Fig. 6C and D). Conversely, Ab-mediated CD8 T-cell deple- / vitro in response to the antigen (Supplementary Data 2). tion in tumor-bearing Lyn mice enhanced tumor burden in þ þ GP100-reactive CD8 T cells were then adoptively transferred bone by BLI, although CD4 depletion did not cause increased / into PLCg2 mice IT injected with B16-FL cells, along with tumor burden (Fig. 6E). To simultaneously measure tumor GP100-pulsed WT DCs and IL-2. Mice injected with IL-2 alone burden and confirm CD4/CD8 depletion, we conducted FACS þ were used as controls. IL-2 itself did not affect tumor growth in analysis on tumor-bearing bones and found that CD8 deple- this model (Supplementary Data 3). Adoptive transfer of tion led to more tumor growth in bone marrow, as determined þ þ GP100-reactive CD8 T cells reduced tumor burden in by TRP-1 staining, a surface marker of B16 cells (Fig. 6F).

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A B Figure 6. T-cell transfer protects 7,000 whereas T-cell depletion Vehicle +Irradiated tumor cells enhances tumor growth in bone of * 0.3 / / 6,000 PLCg2 and Lyn mice, ** 5,000 respectively. A, IFN-g production was measured in splenocytes 4,000 0.2 from tumor-bearing WT, T cells + PLCg2/, and Lyn/ mice 3,000 restimulated with irradiated B16- /IFN γ

+ in vitro IFN- γ (pg/mL) 2,000 0.1 * FL cells . IFN-g levels were PLC 2/

% GP100 specific reduced in g mice and 1,000 CD8 increased in Lyn / mice (*, P < ** / 0 0 0.05 Lyn vs. WT; **, P < 0.01 γ –/– –/– –/– –/– WT PLC 2 Lyn WT PLCγ2 Lyn PLCg2 / vs. WT; n ¼ 3). B, 400 C D percentage of GP100-specific WT CD8þ/IFN-g–producing T cells PLCγ2–/– 0.8 300 PLCγ2–/– was determined by FACS in WT PLC 2/ + T cell spleen of , g ,and 0.6 Lyn/ tumor-bearing mice. 200 * T-cell activation was reduced in photons/s)

4 * 0.4 / Total flux Total PLCg2 mice, whereas it was Lyn/ ( × 10 100 increased in animals 0.2 (*, P < 0.05 PLCg2 / vs. WT;

Tumor area/total area Tumor **, P < 0.01 Lyn / vs. WT; n ¼ 3). 0 0 8 10 (days) WT – T cell + T cell C and D, BLI and histology PLCγ2–/– showed decreased bone tumor burden in PLCg2/ mice EFWT WT –/– adoptively transferred with Lyn + anti-CD8 100 þ PLC 2/ 600 Lyn–/– + anti-CD4 CD8 T cells versus g * / Lyn–/– + IgG controls (*, P < 0.05 vs. PLCg2 80 mice; n ¼ 5). E and F, BLI and 400 60 percentage of B16-FL cells in * bone, determined by FACS using a-TRP-1 Ab to detect B16 cells, photons/s) 40

Total flux Total / 200 cells in BM was done in Lyn mice injected % TRP-1–positive ( × 10 20 with IgG control (CTR), a-CD8, or a-CD4 Abs. Data showed that 0 0 CD8þ T-cell depletion enhanced 8 10 (days) WT α-IgG α-CD8 α-CD4 tumor growth in bone of Lyn/ –/– Lyn mice (*, P < 0.05 a-CD8 vs. a-IgG Ab; n ¼ 5).

þ Thus, CD8 T cells contributed to the regulation of tumor cacy of antiresorptive therapies. By using immunocompetent / / growth in bone in PLCg2 and Lyn mice regardless of OC mice, we showed that alterations in T-cell functions can functionality. bypass the requirement for OCs in tumor growth within bone and reduce the efficiency of ZOL treatment. Discussion PLCg2 plays a critical role in bone homeostasis and immune function (12, 13, 15, 23, 28–30). Previous studies using a tumor- Animal studies using human breast cancer cells injected in rejection assay, in which MHCI–deficient susceptible target / immunocompromised mice strongly support a tumor/bone cells were injected intraperitoneally in WT or PLCg2 mice, / vicious cycle model in which blockade of OC function can suggested compromised tumor elimination in PLCg2 mice reduce tumor growth in bone and prevent associated bone (31). Consistent with this finding, we observed increased s.c. loss (26, 27). ZOL, a potent inhibitor of OC activity, is widely tumor growth and elevated metastatic dissemination follow- used in the clinic to treat patients with bone metastases. ZOL ing intravenous injection of B16-FL cells, showing broadly confers protection from tumor-induced bone loss and reduces impaired antitumor immune responses (Supplementary Data skeletal complications such as bone pain, pathologic fractures, 4). However, in bone, the OC resorptive activity is thought to bone surgery, and hypercalcemia. Unfortunately, one third of be central in regulating metastatic dissemination and tumor patients with bone metastases who respond to ZOL treatment burden (1). By measuring direct tumor growth in bone or / develop further skeletal-related events within 2 years of initi- tropism of tumor cells to bone, we show that PLCg2 OC ating the antiresorptive therapy (5). Therefore, the tumor/ defect is not sufficient to prevent bone metastases, thus bone vicious cycle model must be revisited to include other challenging the current tumor/bone vicious cycle model. bone marrow–derived cells, in addition to the OCs that can Mirroring the PLCg2 / phenotype, deletion of Lyn,a participate in restraining or encouraging the expansion of negative regulator of PLCg activity, leads to increased immune tumor in the bone microenvironment, thus altering the effi- activation (18, 19, 32, 33). In agreement with a hyperactive

4806 Cancer Res; 71(14) July 15, 2011 Cancer Research

Contribution of CD8þ T Cell in Bone Tumor Burden

/ þ / immune condition, Lyn mice are protected from s.c. tumor CD8 T cells was also enhanced in Lyn mice, although this / / growth and metastatic dissemination. Lyn mice are also is barely detectable in PLCg2 animals. Importantly, deple- þ / more prone to bone loss due to increased OC number (20). tion of CD8 T cells restores tumor growth in bone in Lyn þ Although more numerous OCs are expected to create a highly mice, whereas adoptive transfer of tumor-specific WT CD8 T / favorable environment for tumor growth in bone, our data cells into PLCg2 mice reduces bone tumor burden. show that increased immune activation is sufficient to coun- Despite not being expressed in T cells, both PLCg2 and Lyn teract the OC-mediated pro-tumor effect, thus protecting indirectly govern T-cell activation by modulating myeloid cell / Lyn mice from bone metastases. functions. Defective DC-mediated antigen presentation (13, 23) Immune cells have emerged as significant regulators of probably underlies the reduced number of tumor-specific þ / primary cancer development as well as metastasis into ectopic CD8 T cell in PLCg2 mice. Because of impaired DC / tissues. Tumor antigens are presented to naïve T cells, leading functions, PLCg2 mice are protected from antigen-induced þ þ to activation of CD4 and CD8 T cells. Subsequently, tumor- arthritis, an inflammatory condition strongly dependent on T- specific T cells home to tumor sites, where they kill antigen- cell activation (23). It is very likely that DC defects impair T-cell / positive tumor cells (34). However, tumor cells can also escape activation in PLCg2 tumor-bearing mice, thus enhancing the immune system, developing the ability to grow in an bone tumor burden even in the absence of functional OCs. An / immunologically intact host. Several factors released by tumor additional explanation for the tumor phenotype in PLCg2 cells can directly or indirectly induce T-cell immune suppres- mice could be defective NK-cell cytotoxicity (12). It is unlikely sion. TGF-b, released by OCs (35), has positive proliferative that NK-cell abnormalities are primarily responsible for effects on tumor cells but also very potent anti-inflammatory increased tumor growth in these mice, because NK-cell deple- activity (7). Thus, the antitumor effects of OC blockade and/or tion in WT mice can only slightly increase bone tumor burden / inhibition of TGF-b pathway could, at least in part, depend on without reaching the levels observed in PLCg2 animals / interference with T-cell–mediated antitumor responses. (Supplementary Data 5). Conversely, Lyn mice have Furthermore, we showed that global T-cell deficiency expanded macrophage and DC populations (18, 36) as well increases, whereas T-cell activation protects from, growth as hyperactive myeloid cells that augment T-cell responses and þ þ of B16-FL cells in bone. CD8 , and to a lesser extent CD4 , IFN-g production (19). Indeed, we found that tumor-bearing / þ T cells have antitumor capabilities. However, T-cell manip- Lyn mice display increased tumor-antigen–specific CD8 T ulations do not seem to affect the capacity of the cancer cells cells in spleen compared with WT. Therefore, similar to PLCg2 to reach bone, presumably because T-cell activation occurs deficiency, absence of Lyn in the myeloid population is likely to after the cells have arrived in the bone microenvironment. be responsible for enhanced T-cell antitumor responses. þ Importantly, we found that tumor growth in bone in CD8 To the best of our knowledge, this report is the first to T-cell–depleted mice occurs independently of ZOL-mediated analyze the relative contribution of bone and immune cells in OC blockade. This finding is in apparent contradiction with development of bone metastases. We now provide compelling previous reports showing that ZOL reduces bone tumor evidence that a condition of immune deficiency can interfere burden in immunocompromised animals. However, a direct with the antitumor effects of OC blockade. We used both comparison between the antitumor effect of ZOL in immu- genetic models of immune and OC modulation (Lyn and nocompetent and immunodeficient mice has never been PLCg2), as well as pharmacologic inhibition of OCs and T reported. ZOL is effective in decreasing tumor burden in T- cells. These findings suggest that the immune status can affect cell–deficient mice compared with untreated immunodefi- the efficacy of antiresorptive treatments in reducing bone þ cient controls. However, the antitumor effect of ZOL in CD8 metastases and emphasize the need to further explore the T-cell–depleted mice is significantly reduced compared with beneficial effects of combined T-cell stimulation and immunocompetent WT animals treated with the antiresorp- antiresorptive therapies in patients with bone metastases. tive agent. This observation has important clinical relevance because ZOL is the current treatment of choice for patients Disclosure of Potential Conflicts of Interest with bone metastases. Although ZOL has been shown to successfully reduce incidence of bone metastases, not all No potential conflicts of interest were disclosed. patients respond well (5). Considering that cancer patients Acknowledgments often have immune imbalances, our data suggest that reduced T-cell antitumor immune responses could be responsible for We gratefully thank Tonia Thompson for research administration and Karon tumor growth in bone despite ZOL-mediated OC blockade. Hertlein for secretarial support (Department of Orthopaedics, Washington Altered T-cell activation is also central to the tumor/bone University). This work was supported by NIH to R. Faccio (grant no. RO1 PLC 2/ Lyn/ AR53628) and ARRA to R. Faccio (grant no. 63181), NIH to D.V. Novack (grant phenotype observed in g and mice. Although nos. AR052705 and EB007568), A.C. Hirbe (grant no. T32HL007088), and K. B16 is a poorly immunogenic cell line, it has been reported Weilbaecher (grant no. R01 52152) and the Barnes-Jewish Foundation to D.V. that it can induce a modest antitumor T-cell response (9). We Novack. þ The costs of publication of this article were defrayed in part by the further confirmed this observation by identifying CD8 T cells payment of page charges. This article must therefore be hereby marked that can respond to the endogenous B16 antigen, GP100. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this þ fact. Specifically, we observed enhanced GP100-specific CD8 T / cells in tumor-bearing Lyn mice but a blunted response in / Received October 28, 2010; revised April 6, 2011; accepted May 12, 2011; PLCg2 animals. Release of IFN-g by these tumor-specific published OnlineFirst May 20, 2011.

www.aacrjournals.org Cancer Res; 71(14) July 15, 2011 4807

Zhang et al.

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4808 Cancer Res; 71(14) July 15, 2011 Cancer Research

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2011 American Association for Cancer Research. Cancer Correction Research Correction: CD8þ T Cells Regulate Bone Tumor Burden Independent of Osteoclast Resorption

In this article (Cancer Res 2011;71:4799–808), which was published in the July 15, 2011, issue of Cancer Research (1), the author listing was incomplete. Speciﬁcally, the listing was missing an institution. In addition, a funding source and grant number were missing from the Acknowledgments section. The correct author listing and missing acknowledgments are provided. The authors regret these errors.

Kaihua Zhang1, Seokho Kim1, Viviana Cremasco1, Angela C. Hirbe2, Lynne Collins3, David Piwnica-Worms3, Deborah V. Novack4, Katherine Weilbaecher2, and Roberta Faccio1

Departments of 1Orthopedics, 2Molecular Oncology, 3BRIGHT Institute, Mallinck- rodt Institute of Radiology and Department of Cell Biology & Physiology, and 4Department of Medicine, Washington University School of Medicine, St. Louis, Missouri

Acknowledgments This work was also supported by NIH to David Piwnica-Worms (grant no. P50CA94056). The histologic and mCT analysis was supported by The Center for Musculoskeletal Biology and Medicine, Award Number P30AR057235 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases.

Reference 1. Zhang K, Kim S, Cremasco V, Hirbe AC, Collins L, Piwnica-Worms D, et al. CD8þ Tcells regulate bone tumor burden independent of osteoclast resorption. Cancer Res 2011;71: 4799–808.

Published online January 17, 2012. doi: 10.1158/0008-5472.CAN-11-3840 Ó2012 American Association for Cancer Research.

568 Cancer Res; 72(2) January 15, 2012 Published OnlineFirst May 20, 2011; DOI: 10.1158/0008-5472.CAN-10-3922

CD8+ T Cells Regulate Bone Tumor Burden Independent of Osteoclast Resorption

Kaihua Zhang, Seokho Kim, Viviana Cremasco, et al.

Cancer Res 2011;71:4799-4808. Published OnlineFirst May 20, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-10-3922

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