Published OnlineFirst January 11, 2012; DOI: 10.1158/0008-5472.CAN-11-3380
Cancer Microenvironment and Immunology Research
VEGF Receptor Inhibitors Block the Ability of Metronomically Dosed Cyclophosphamide to Activate Innate Immunity–Induced Tumor Regression
Joshua C. Doloff and David J. Waxman
Abstract In metronomic chemotherapy, frequent drug administration at lower than maximally tolerated doses can improve activity while reducing the dose-limiting toxicity of conventional dosing schedules. Although the antitumor activity produced by metronomic chemotherapy is attributed widely to antiangiogenesis, the significance of this mechanism remains somewhat unclear. In this study, we show that a 6-day repeating metronomic schedule of cyclophosphamide administration activates a potent antitumor immune response associated with brain tumor recruitment of natural killer (NK) cells, macrophages, and dendritic cells that leads to marked tumor regression. Tumor regression was blocked in nonobese diabetic/severe combined immunodeficient (NOD/SCID-g) mice, which are deficient or dysfunctional in all these immune cell types. Furthermore, regression was blunted by NK cell depletion in immunocompetent syngeneic mice or in perforin-deficient mice, which are compromised for NK, NKT, and T-cell cytolytic functions. Unexpectedly, we found that VEGF receptor inhibitors blocked both innate immune cell recruitment and the associated tumor regression response. Cyclophosphamide administered at a maximum tolerated dose activated a transient, weak innate immune response, arguing that persistent drug-induced cytotoxic damage or associated cytokine and chemokine responses are required for effective innate immunity–based tumor regression. Together, our results reveal an innate immunity–based mechanism of tumor regression that can be activated by a traditional cytotoxic chemotherapy administered on a metronomic schedule. These findings suggest the need to carefully evaluate the clinical effects of combination chemotherapies that incorporate antiangiogenesis drugs targeting VEGF receptor. Cancer Res; 72(5); 1103–15. 2012 AACR.
Introduction chemotherapy also induces the antiangiogenic glycoprotein Maximum tolerated dose (MTD) chemotherapy has been a thrombospondin-1 (TSP1; Thbs1; refs. 4, 5), suggesting that mainstay in the cancer clinic for the past 50 years. However, antiangiogenesis is an important factor in the superior anti- fi recent preclinical successes with metronomic chemotherapy, tumor pro les of metronomic regimens (6, 7). However, this fi where drug is administered at a regular, more frequent interval, proposed mechanism is not supported by the nding that bona fi but at a lower dose than MTD chemotherapy, have been rapidly de antiangiogenic drugs often show only moderate antitumor translated into clinical trials, where improved antitumor activity when used as single agents, despite their effectiveness responses have been observed (1). Metronomic chemotherapy at inhibiting tumor angiogenesis. Examples of this include – eliminates the need for extended recovery periods between non small cell lung cancer (8) and glioblastomas (9) in human treatment cycles and thus allows for persistent drug treatment patients, 9L gliosarcoma xenografts treated in severe com- fi in a way that minimizes drug toxicity to the patient (2). bined immunode cient (SCID) mice (4), and metastatic mel- Metronomic chemotherapy schedules using cyclophospha- anomas in C57BL/6 mice (10). Thus, other mechanisms for the mide and other chemotherapeutic drugs induce endothelial improved antitumor effects of metronomic chemotherapy are cell death in addition to tumor cell death (2–4). Metronomic likely operative. TSP1, in addition to its antiangiogenic activity, has other actions, including stimulation of chemotaxis, cell proliferation, Authors' Affiliation: Division of Cell and Molecular Biology, Department of and protease regulation in healing (11). Moreover, tumors that Biology, Boston University, Boston, Massachusetts stably express TSP1 have significantly increased levels of fi Note: Supplementary data for this article are available at Cancer Research in ltrating antitumor M1 macrophages (12), suggesting a role Online (http://cancerres.aacrjournals.org/). for the host immune system in the improved tumor responses Corresponding Author: David J. Waxman, Department of Biology, Boston to metronomic drug treatments. University, 5 Cummington Street, Boston, MA 02215. Fax: 1-617-353- Presently, we show that a 6-day repeating metronomic 7404; E-mail: [email protected] schedule of cyclophosphamide activates a potent and sus- doi: 10.1158/0008-5472.CAN-11-3380 tained antitumor innate immune response that is associated 2012 American Association for Cancer Research. with tumor regression and leads to ablation of large brain
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tumor xenografts. In contrast, MTD cyclophosphamide treat- tumor volume set to 100%) once average tumor volumes ment induces a weak immune response that dissipates during reached 500 mm3. Mice were treated with cyclophospha- the rest period between treatment cycles. We further show that mide given on an intermittent metronomic schedule (140 mg antitumor innate immunity, and not antiangiogenesis, is the cyclophosphamide/kg body weight, repeated every 6 days) major mechanism for the marked tumor regression seen in or on an MTD schedule (150 mg cyclophosphamide/kg these models. Supporting this hypothesis, tumor regression body weight on each of 2 consecutive days, followed by a is blocked in natural killer (NK) cell–deficient and macro- 19-day rest period) as indicated on each figure using vertical phage and dendritic cell dysfunctional nonobese diabetic/ arrows. Axitinib and AG-028262 were administered daily at SCID (NOD/SCID-g) mice (13) and is blunted by NK cell 25 mg/kg body weight/d intraperitoneally and cediranib at depletion in an immunocompetent syngeneic mouse model 5 mg/kg body weight/d intraperitoneally for up to 24 days, as and in mice deficient in the lymphocyte effector molecule indicated in each study. NK cell–depleting monoclonal perforin, where NK, NKT, and T-cell cytolytic function are antibody anti-asialo-GM1 (cat# 986–10001, Wako Chemicals compromised (14). USA) was administered intraperitoneally at a dose of 50 mL In addition, we show that VEGF receptor–selective antian- (diluted 1:3 in sterile 1PBStofinal volume of 150 mLfor giogenic drugs block antitumor immunity and prevent met- each mouse on the day of injection) and delivered once every ronomic cyclophosphamide–induced tumor regression. VEGF 6daysstarting3dayspriortothefirst metronomic cyclo- receptor signaling is important for dendritic cell–endothelial phosphamide treatment (i.e., asialo-GM1 antibody was given cell cross-talk, transdifferentiation (15), tumor-associated 3 days prior to each cyclophosphamide injection). On com- macrophage infiltration (16), and chemokine expression and bination therapy days, cyclophosphamide was administered secretion in proinflammatory responses (17). Furthermore, 4 hours prior to treatment with a VEGF receptor inhibitor to endothelial cells and immune cells have shared bone mar- minimize the potential for drug interactions. Tumor sizes row–derived stem and progenitor cells regulated by VEGF and mouse body weights were measured at least twice a receptor (18), suggesting that compounds designed to kill week. Tumor growth rates prior to drug treatment were tumor blood vessels by inhibiting VEGF receptor signaling similar among all normalized groups. may also elicit immunosuppressive responses. Quantitative PCR and statistical analysis RNA isolation, quantitative PCR (qPCR) primer design, Materials and Methods and qPCR analyses were done as described in Supplemen- Cell lines tary Materials and Methods. qPCR data are expressed as Human U251 glioblastoma cells (National Cancer Institute), mean values SE for n ¼ 5 to 6 individual tumors from 3 rat 9L gliosarcoma cells (Neurosurgery Tissue Bank, University mice per time point per treatment group unless indicated of California, San Francisco), and mouse GL261 glioma cells otherwise. Statistically significant differences between (DCTD, DTP Tumor Repository) were authenticated by and mean values of different treatment groups were determined obtained from the indicated sources. Cells were grown at 37 C by 2-tailed Student t test ( , P < 0.05; , P < 0.001; , in a humidified 5% CO2 atmosphere. U251 and GL261 cells were P < 0.0001). grown in RPMI-1640 and 9L cells in Dulbecco's Modified Eagle's Media, all of which contained 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin. Other methods Sources of reagents and drugs, fluorescence-activated cell Mouse models and tumor xenografts sorting (FACS) analysis, and immunohistochemical methods Five-week-old (24–26 g) male ICR/Fox Chase immunode- are described in Supplementary Materials and Methods. ficient SCID mice (Taconic Farms), 5-week-old male NOD. Cg- Prkdcscid Il2rgtm1Wjl/SzJ (NOD-SCID-g, NSG) mice (The Jackson Results Laboratory), and 5-week-old (22–24 g) male C57BL/6 (wild- Metronomic cyclophosphamide–induced regression of type, immunocompetent; Taconic) and C57BL/6-Prf1 (per- brain tumor xenografts does not involve forin knockout; The Jackson Laboratory) mice were housed antiangiogenesis and treated under approved protocols and federal guidelines. Although antiangiogenesis is considered a key mechanistic Tumor cells (2 106 GL261 glioma cells, 4 106 9L gliosar- feature of metronomic chemotherapy, metronomic cyclophos- coma cells, or 6 106 U251 glioblastoma cells) were injected phamide treatment of SCID mice bearing U251 glioblastoma s.c. on each posterior flank in 0.2 mL serum-free RPMI using a xenografts induces tumor regression that is sustained and 0.5-inch 29-gauge needle and a 1-mL insulin syringe. 9L and complete (Fig. 1A) in the absence of antiangiogenesis U251 tumor xenografts were grown s.c. on the flanks of SCID or (Fig. 1B). In contrast, the VEGF receptor–selective inhibitor NSG mice, and GL261 tumors were inoculated into the flanks of axitinib (19) is strongly antiangiogenic (Fig. 1B) yet primarily C57BL/6 (wild-type or Prf1 ) mice. Tumor areas (length shows a growth static response, followed by rapid tumor width) were measured twice weekly using Vernier calipers regrowth upon discontinuation of treatment (Fig. 1A). Axitinib (VWR, Cat# 62379-531), and tumor volumes were calculated initially increased antitumor activity when combined with on the basis of the formula, Vol ¼ (p/6) (L W)3/2. Tumors metronomic cyclophosphamide treatment but ultimately were monitored and treatment groups were normalized (each blocked the tumor regression seen with metronomic therapy
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Metronomic Cyclophosphamide Activates Innate Immunity, Tumor Regression
A U251 Relative tumor size (%)
B C CD31 protein mTSP1 (9L) mTSP1 (U251)
% area positive area % ** ** Relative expression Relative
D mPEDF (9L) mPEDF (U251) CD31 RNA Relative expression Relative Relative expression Relative
Figure 1. Metronomic cyclophosphamide (CPA)–induced regression of U251 tumors and inhibitory effects of the VEGF receptor–selective inhibitor axitinib (Ax). A, human U251 glioblastoma xenografts grown in SCID mice were untreated (UT) or were treated with metronomic (metro) cyclophosphamide (140 mg/kg intraperitoneally, every 6 days for 17 cycles; arrows along x-axis), daily axitinib (25 mg/kg, intraperitoneally daily for 24 days), or metronomic cyclophosphamide in combination with daily axitinib. Tumor volumes were normalized to 100% on the day of first drug treatment (day 0), when group averages reached about 500 mm3 (n ¼ 12 tumors per group). Tumors were inoculated 32 days prior to the start of drug treatment. B, U251 tumors treated as indicated were immunohistochemically stained for the endothelial cell marker CD31 protein (top; 12 days after initiating drug treatment; , P < 0.001 compared with UT control group) or assayed by qPCR for CD31 RNA (bottom; 6 days after the first, second, and third cyclophosphamide treatments, as marked along the x-axis). CD31 immunohistochemical staining was quantified as described under Supplementary Materials and Methods. qPCR analysis of host (m, mouse) TSP1 (C) and PEDF expression (D) in 9L rat (left) and U251 human (right) tumors grown in SCID mice, treated as in (A) and isolated 6 days after the indicated number of cyclophosphamide injections. For each comparison, qPCR data were normalized to the first untreated (UT) tumor group, whose relative RNA level was set to 1. Where indicated ("m"), qPCR analysis was carried out using mouse (host)-specific primers. Bars, mean SE for n ¼ 5–6 tumors/group. See also Supplementary Fig. S1. PEDF, pigment epithelium–derived factor. alone. Partial tumor regression was eventually seen after Metronomic cyclophosphamide activates antitumor discontinuation of axitinib treatment, followed by rapid tumor innate immunity regrowth after termination of metronomic chemotherapy. An To better understand why metronomic cyclophospha- initial improvement in tumor response followed by inhibition mide fully regresses U251 tumors, even in the absence of of metronomic cyclophosphamide–induced tumor regression antiangiogenesis, we investigated other potential mechan- was also observed with axitinib cotreatment in the 9L glio- isms and found that several macrophage-associated host sarcoma model (4). factors are increased in the metronomic cyclophosphamide–
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treated tumors, including TSP1 (Fig. 1C). While TSP1 has Supplementary Fig. S2A), macrophage cytolytic effectors antiangiogenic activity (5), it has also been linked to tumor lysozyme 1 and 2 (Supplementary Fig. S2B), and the death infiltration by antitumor M1 macrophages (12). Metrono- receptor Fas (Fig. 2B), which activates macrophages and mic cyclophosphamide also increased host (mouse cell) increases their tumor cytotoxicity (21). expression of pigment epithelium–derived factor (PEDF; The above findings suggested that metronomic cyclo- Serpinf1;Fig.1D),anantiangiogenicfactorthatalsostimu- phosphamide activates an innate immune response in the lates M1 macrophage recruitment to tumors (20). These regressing tumors. To investigate this possibility, we con- responses were seen in both rat 9L and human U251 tumors sidered NK cells, given the role of Fas in mediating inter- grown in SCID mice, where TSP1 and PEDF levels were actions between NK cells and cells marked for destruction strongly correlated (r ¼ 0.89) in large sets of untreated and (22). In metronomic cyclophosphamide–treated tumors, we drug-treated tumors isolated various times after initiation observed large increases in the host NK cell marker NK1.1 of metronomic cyclophosphamide treatment (Supplemen- and in the NK cell–associated cytotoxic granzymes A, B, and tary Fig. S1). Metronomic cyclophosphamide also induced C and perforin (Fig. 2A and C; Supplementary Figs. S2C and the host macrophage markers CD68 and F4/80 (Fig. 2A; S3A–S3C), which are essential for cytotoxic lymphocyte–
A CD68 NK1.1 CD74 B No Rx No Rx No Rx mFas
Figure 2. Metronomic cyclophosphamide (Metro CPA) induces and axitinib (Ax) blocks Relative expression Relative recruitment of macrophages, NK UT CPA Ax Ax + cells, and dendritic cells to U251 CPA CPA CPA CPA C tumors. A, immunostaining of mGzmB macrophage (left), NK cell (middle), and dendritic cell markers (right) in U251 tumors, untreated (no Rx) or treated with metronomic cyclophosphamide axitinib and excised on treatment day 12, as
Relative expression Relative shown in Fig. 1A. Representative UT CPA Ax Ax + images are shown with signal CPA + Ax CPA + Ax CPA + Ax CPA intensities equivalent to group mPerforin mean ImageJ quantification data (i. e., Supplementary Fig. S2). B, C, and E, expression of the indicated host factors (m, mouse) in U251 tumors 6 days after the first, 10X second, or third metronomic Relative expression Relative cyclophosphamide injection UT CPA Ax Ax + axitinib treatment, as marked. CPA qPCR data were normalized to the D Untreated Metro CPA Metro CPA + Ax first untreated (UT) tumor group,
+ whose relative RNA level was set to 2.32% 11.87% 1.86% 1. Bars, mean SE for n ¼ 5–6 tumors/group. D, FACS analysis of þ NK1.1 NK1.1 cells (%) in single-cell suspensions prepared from untreated and cyclophosphamide- treated and/or axitinib-treated 9L tumors grown in SCID mice. mCD207 (Langerin) mCD209 (DC-SIGN) E Immunoglobulin G background control, 0.54%. CD49b (no change with treatment) was the marker along the x-axis. See also Supplementary Fig. S2. Relative expression Relative Relative expression Relative
UT CPA Ax Ax + UT CPA Ax Ax + CPA CPA
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Metronomic Cyclophosphamide Activates Innate Immunity, Tumor Regression
mediated cell death (22). FACS analysis confirmed the influx (25), were also increased in the metronomic cyclophospha- þ of NK1.1 cells into the metronomic cyclophosphamide– mide–treated U251 and 9L tumors (Fig. 3A and B), suggest- treated tumors (Fig. 2D; Supplementary Fig. S3D). Dendritic ing their role in mobilizing the host innate immune cells were also recruited to the tumors, as shown by the response. activation of host dendritic cell markers important for cell– target interactions, and antigen presentation to the adaptive VEGF receptor–targeted inhibitors block metronomic immune system (Fig. 2E). CD74, implicated in the regulation cyclophosphamide–activated antitumor immunity of dendritic cell migration (23), was also increased (Fig. 2A; We next sought to determine why the VEGF receptor Supplementary Figs. S2D and S3A and S3B). Because NK1.1 inhibitor axitinib blocks tumor regression induced by metro- and dendritic cell markers are both increased in the cyclo- nomic cyclophosphamide treatment (Fig. 1A; ref. 4). In both þ phosphamide-treated tumors, other NK1.1 cells, such as U251 and 9L tumors, axitinib blocked tumor infiltration of NK IFN-producing killer dendritic cells (24), could also be cells, macrophages, and dendritic cells (Fig. 2; Supplementary involved. Host cytokine and chemokine immune attractants, Figs. S2 and S3A–S3C). Axitinib also blocked the formation of such as interleukin (IL)-12b and CXCL14, which can influ- chemokine and cytokine gradients that may mobilize these ence leukocyte and lymphocyte activation and migration cells into tumors (Fig. 3A and B). Importantly, axitinib
A mIL-12β (U251) mIL-12β (9L)
Figure 3. Axitinib (Ax) blocks Relative expression
cytokine, chemokine, and other Relative expression responses to metronomic cyclophosphamide (CPA). qPCR UT 12444 analysis of host (m, mouse) UT CPA Ax Ax + b CPA Ax Ax + macrophage-associated IL-12 (A), CPA NK cell–associated CXCL14 (B), and CPA human tumor cell–specific MICB, an B activating ligand for the immune cell mCXCL14 (U251) mCXCL14 (9L) receptor NKG2D (C), in metronomic cyclophosphamide–treated U251 and 9L tumor xenografts grown in SCID mice. D, axitinib, both alone and in combination with metronomic cyclophosphamide, shifted the tumor-infiltrating macrophage subpopulations to the protumor M2
subtype, as indicated by the ratio of Relative expression protumor (M2) macrophage marker Relative expression (arginase-1; Arg1) to antitumor (M1) macrophage marker [inducible nitric UT 12444 UT CPA Ax Ax + oxide synthase (iNOS); Nos2] CPA Ax Ax + CPA expression in the same set of U251 CPA tumors. qPCR data were normalized fi C D to the rst untreated (UT) tumor Human NKG2D ligand MICB (U251) mArg1/mNos2 group, whose relative RNA level was (M2/M1) set to 1. Bars, mean SE for n ¼ 5–6 tumors/group. Relative expression Relative expression
UT CPA Ax Ax + UT CPA Ax Ax + CPA CPA
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B Figure 4. Immunosuppressive A Endothelial marker CD31 Granzyme B effects of VEGF receptor tyrosine kinase inhibitors (RTKI). U251 tumors grown in SCID mice were treated with metronomic cyclophosphamide (CPA) given alone or in combination with daily axitinib (Ax, 25 mg/kg/d, intraperitoneally), cediranib (AZD, AZD2171; 5 mg/kg/d, intraperitoneally), or AG-028262 Relative expression Relative expression (AG, 25 mg/kg/d, intraperitoneally). Each antiangiogenic drug was UT CPA Ax AZD AG UT CPA Ax AZD AG given daily for 18 days prior to the – CPA CPA analysis shown. A, VEGF receptor selective inhibitors suppress tumor angiogenesis (CD31 expression) C RTKI immune knockdown screen (U251) and (B) block metronomic cyclophosphamide induction of granzyme B in U251 tumors grown in SCID mice, as compared with untreated (UT) controls, determined 6 days after the third cyclophosphamide injection (day 18). C, VEGF receptor–selective inhibitors block metronomic cyclophosphamide–induced immune recruitment in U251 tumors, as determined by qPCR analysis of NK cell markers NKp46 and NK1.1, macrophage marker
Relative expression CD68, and dendritic cell markers CD74 and CD209. qPCR data were normalized to untreated tumors, whose relative RNA level was set to 1. Bars, mean SE for n ¼ 5–6 UT CPA Ax AZD AG tumors/group. CPA
suppressed these immune factors to basal or below basal levels. AG-028262 strongly inhibited tumor angiogenesis, as expected Axitinib also suppressed the induction by metronomic cyclo- (Fig. 4A); however, they also blocked metronomic cyclophos- phosphamide of human tumor cell–expressed MICB (Fig. 3C), phamide–induced NK cell activity (Fig. 4B) and the recruit- which is an activating ligand for the receptor NKG2D found on ment of all 3 classes of innate immune cells (Fig. 4C). More- NK and other immune cells (26). This finding suggests that over, by the sixth day of treatment, both drugs blocked axitinib might further inhibit metronomic cyclophosphamide– U251 regression induced by metronomic cyclophosphamide, induced tumor cell–targeted antitumor immunity by blocking resulting in tumor growth stasis that continued until the study the expression and subsequent presentation of activation was terminated on treatment day 18, whereas over that same signals by cyclophosphamide-damaged tumor cells. Finally, time period, tumors treated with metronomic cyclophospha- axitinib shifted the balance oftumor-associatedmacro- mide alone regressed a further 50% in volume. Thus, interfer- phages from antitumor M1 [marked by inducible nitric oxide ence with metronomic chemotherapy–induced antitumor synthase (iNOS)] to protumor M2 macrophages (marked by immunity and tumor regression are general responses to VEGF arginase-1; Fig. 3D). Thus, the inhibitory effects of axitinib on receptor inhibitors. metronomic cyclophosphamide–induced tumor regression likely result from interference with the innate immune Metronomic cyclophosphamide–induced tumor response at multiple levels. regression requires innate immune cells Next, we used 2 other antiangiogenic tyrosine kinase inhi- To test whether the tumor-infiltrating innate immune cells bitors, cediranib (AZD2171; ref. 27) and AG-028262 (28), to contribute functionally to tumor regression, we investigated investigate the importance of VEGF receptor signaling for the effects of metronomic cyclophosphamide treatment on metronomic cyclophosphamide–induced antitumor immuni- 9L gliosarcomas grown in NOD-SCID-IL2Rg-null (NSG) mice, ty. These 2 chemicals exhibit selectivity for VEGF receptor which, unlike SCID mice, are NK cell deficient and have inhibition comparable with or greater than that of axitinib dysfunctional macrophages and dendritic cells due to loss of (27, 28). In U251 tumors grown in SCID mice, cediranib and the important immunostimulatory IL-2Rg receptor (13). In
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Metronomic Cyclophosphamide Activates Innate Immunity, Tumor Regression
A B 9L (SCID), UT 0 CPA (d0) 0 CPA (d18) 9L (NSG), UT 2 CPA (d12) 4 CPA (d24) 7 CPA (d42)
Figure 5. Antitumor activity of metronomic cyclophosphamide 9L (NSG) + CPA (CPA) in NSG mice (adaptive and innate immunocompromised). A, 9L tumor growth profiles in NSG and Relative tumor size (%) 9L (SCID) + CPA Fold change over day 0 SCID mice, with tumors implanted 14 days prior to the first (DC) (Platelets) (Neutrophils) cyclophosphamide treatment (n ¼ 12 Days tumors/group). B, qPCR of the indicated factors in 9L tumors C Tumor Spleen 10 1.0 metronomic cyclophosphamide NKp46 NKp46 treatment and isolated on day 0 or 6 8 0.8 days after the second, fourth, or 6 0.6 seventh treatment cycle (days 12–42, 4 0.4 as marked). Bars, mean SE for 2 0.2 n ¼ 5–6 tumors/group. C, qPCR of ND 0.0 the indicated factors in 9L tumors 0 NK1.1 NK1.1 (left) and spleens (right) 1.00 6 metronomic cyclophosphamide 0.75 treatment isolated from NSG or SCID 4 mice on day 0 or 6 days after the 0.50 second, fourth, or seventh treatment 2 0.25 cycle. For each comparison, qPCR 0 0.00 data were normalized to the first 40 GzmB GzmB untreated (UT) SCID tumor or spleen 1.00 30 group, whose relative RNA level was 0.75 set to 1. The absence of NKp46, 20 0.50 granzyme B (GzmB), and perforin 10 fl fi 0.25 (Prf1) re ects the NK cell de ciency ND of NSG mice. See Supplementary Relative expression 0 0.00 Prf1 Prf1 Materials for CT values determined 70 1.0 by qPCR. White bars, untreated 0.8 50 tumor and spleen samples; shaded 0.6 bars, treated samples. Bars, mean 30 0.4 ¼ – 0.2 SD for tumor (n 4 6) and spleen 10 ND ND (n ¼ 2–3) pools. DC, dendritic cell; 0 0.0 CD68 ND, not detectable. 10 CD68 8 1.0 6 4 0.5 2 0 0.0 03247 2 3324 03247 2 3324 UT CPA UT CPA UT CPA UT CPA 9L, NSG 9L, SCID 9L, NSG 9L, SCID
NSG mice, metronomic cyclophosphamide–induced 9L tumor phamide-treated NSG spleens as compared with SCID mice growth delay led to growth stasis after several treatment cycles (Fig. 5C, right: NSG vs. SCID), as expected. Progressive deple- but no regression (Fig. 5A). While macrophages, dendritic cells, tion of NK cells from SCID mouse splenic reservoirs with and markers for other innate immune cell types such as continued metronomic cyclophosphamide treatment was also neutrophils and platelets are present in the tumors and are apparent (Fig. 5C, right). The tumor growth stasis seen in NSG increased by metronomic cyclophosphamide (Fig. 5B and C), mice likely reflects residual antitumor immune cell responses NK cells (NKp46) and their cytotoxic effectors, granzyme B and (Fig. 5B and C) and the intrinsic cytotoxicity of cyclophospha- perforin, were undetectable (Fig. 5C). Thus, the large differen- mide toward tumor cells and tumor endothelial cells. The tial antitumor response between NSG mice (tumor growth delayed increase in the NK cell markers NKp46 and perforin in stasis) and SCID mice (full tumor regression; Fig. 5A) can be cyclophosphamide-treated SCID mouse 9L tumors (Fig. 5C) is attributed to the diminished innate immune response to consistent with the delayed onset of tumor regression (Fig. 5A), metronomic cyclophosphamide in NSG mice (Fig. 5C, left: further implicating NK cell function in this response to met- NSG vs. SCID). NK cell factors (NKp46, NK1.1, GzmB, and Prf1) ronomic cyclophosphamide. Finally, a strong increase in TSP1 were also deficient in spleen in both untreated and cyclophos- expression was seen in the metronomic cyclophosphamide–
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– Fold change over day 0 uosgop e loSplmnayFg S3. Fig. Supplementary also See tumors/group. 6