Chemotherapy induces enrichment of CD47+/CD73+/PDL1+ immune evasive triple-negative breastcancer cellsDebangshu Samantaa,b, Youngrok Parka,b, Xuhao Nic,d, Huili Lic, Cynthia A. Zahnowc, Edward Gabrielsonc,e, Fan Panc,d,and Gregg L. Semenzaa,b,c,d,f,g,h,1

aMcKusick–Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205; bJohns Hopkins Institute for CellEngineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; cDepartment of Oncology, Johns Hopkins University School of Medicine,Baltimore, MD 21205; dDepartment of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205; eDepartment of Pathology, JohnsHopkins University School of Medicine, Baltimore, MD 21205; fDepartment of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205;gDepartment of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and hDepartment of Biological Chemistry, JohnsHopkins University School of Medicine, Baltimore, MD 21205

Contributed by Gregg L. Semenza, December 21, 2017 (sent for review October 18, 2017; reviewed by Sean P. Colgan and Michail V. Sitkovsky)

Triple-negative breast cancer (TNBC) is treated with cytotoxicchemotherapy and is often characterized by early relapse andmetastasis. To form a secondary (recurrent and/or metastatic)tumor, a breast cancer cell must evade the innate and adaptiveimmune systems. CD47 enables cancer cells to evade killing bymacrophages, whereas CD73 and PDL1 mediate independentmechanisms of evasion of cytotoxic T lymphocytes. Here, wereport that treatment of human or murine TNBC cells withcarboplatin, doxorubicin, gemcitabine, or paclitaxel induces thecoordinate transcriptional induction of CD47, CD73, and PDL1mRNA and protein expression, leading to a marked increase inthe percentage of CD47+CD73+PDL1+ breast cancer cells. Geneticor pharmacological inhibition of hypoxia-inducible factors (HIFs)blocked chemotherapy-induced enrichment of CD47+CD73+PDL1+

TNBC cells, which were also enriched in the absence of chemother-apy by incubation under hypoxic conditions, leading to T cell anergyor death. Treatment of mice with cytotoxic chemotherapy markedlyincreased the intratumoral ratio of regulatory/effector T cells, aneffect that was abrogated by HIF inhibition. Our results delineatean HIF-dependent transcriptional mechanism contributing to TNBCprogression and suggest that combining chemotherapy with an HIFinhibitor may prevent countertherapeutic induction of proteins thatmediate evasion of innate and adaptive antitumor immunity.

HIF-1 | effector T cells | regulatory T cells | adenosine | PD-1

Triple-negative breast cancers (TNBCs), which lack expressionof the estrogen receptor, progesterone receptor, and HER2,

comprise ∼15% of all breast cancers (1). Targeted therapy is notavailable for TNBC, and cytotoxic chemotherapy is the primarysystemic treatment (2). After an initial response to chemotherapy,many patients with TNBC have recurrent disease, which is drug-resistant and metastatic, leading to increased mortality comparedwith other breast cancer subtypes (1, 2). Thus, to reduce patientmortality, it is critical to understand the properties of TNBC cellsthat survive chemotherapy.The ability of cancer cells to evade both the innate and adaptive

immune systems plays a critical role in cancer relapse and metas-tasis (3–5). In breast cancer, the frequency of intratumoral immuneinfiltrates is significantly decreased in invasive breast cancer ascompared with ductal carcinoma in situ (6). Among patients withTNBC, the absence of intratumoral immune infiltrates is associatedwith patient mortality (7). The aggressive nature of the recurrent,metastatic disease that follows chemotherapy in patients withTNBC suggests a further loss of antitumor immunity, which issupported by limited in vitro studies (8), but the underlying mo-lecular mechanisms have not been established.Several studies have implicated hypoxia-inducible factor 1α

(HIF-1α) in the regulation of innate and adaptive immunity.

Conditional knockout (cKO) of HIF-1α in the granulocyte-monocyte lineage impaired inflammatory responses by thesecells (9). Defective B cell development and autoimmunity werefound in chimeric KO mice lacking expression of HIF-1α in Band T lymphocytes (10). HIF-1α cKO in T cells was associatedwith increased levels of proinflammatory cytokines and aug-mented antibacterial immunity (11). HIF-1α cKO selectively inCD8+ T cells led to impaired tumor infiltration and tumor cellkilling (12). HIF-1α cKO selectively in CD4+ T cells led to de-creased regulatory T (Treg) cell antiinflammatory effects in models ofautoimmune encephalitis (13) and colitis (14). However, thesystemic effect of pharmacological inhibition of HIF-1 activityon antitumor immunity has not been determined.A major mechanism mediating evasion of innate immunity by

cancer cells is the expression of CD47, a cell-surface protein thatinteracts with signal regulatory protein α on macrophages to blockphagocytosis (15–17). HIF-1 stimulates CD47 expression in hyp-oxic breast cancer cells (18). However, whether chemotherapy alsoaffects the expression of CD47 has not been investigated.Tumor cells escape from adaptive immunity by altering the

expression of the B7 family of costimulatory ligands, whichmodulate effector T (Teff) cell responses (19, 20). There hasbeen great interest in the programmed cell death-1 (PD1)receptor, which is expressed on T cells and interacts with its

Significance

Cytotoxic chemotherapy is frequently used in patients withtriple-negative breast cancer (TNBC). Although patients initiallyrespond to the treatment, the cancer often comes back and killsthe patient. Recent studies have demonstrated that cancer cellsexpress genes that protect them from killing by immune cells,but the stimulus that prompts this response is unknown. Weshow that when TNBC cells are treated with chemotherapy, thesurviving cells turn on genes that enable them to escape killingby the immune system. We identify hypoxia-inducible factors(HIFs), which are known to promote metastasis of TNBC, asresponsible for this countertherapeutic effect. We show thatcoadministration of an HIF inhibitor with chemotherapy blocksthe ability of surviving TNBC cells to evade the immune system.

Author contributions: D.S. and G.L.S. designed research; D.S., Y.P., X.N., H.L., C.A.Z., E.G., andF.P. performed research; D.S. and G.L.S. analyzed data; and D.S. and G.L.S. wrote the paper.

Reviewers: S.P.C., University of Colorado Health Sciences Center; and M.V.S., NortheasternUniversity.

The authors declare no conflict of interest.

Published under the PNAS license.1To whom correspondence should be addressed. Email: gsemenza@jhmi.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1718197115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1718197115 PNAS | Published online January 24, 2018 | E1239–E1248

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cognate ligand PDL1 (also known as CD274) on cancer cells(21). Remarkable therapeutic responses to anti-PD1 or anti-PDL1 antibodies have occurred in a minority of patients withmelanoma, non-small cell lung cancer, and renal carcinoma (22,23). However, these immunotherapies are neither effectiveagainst all cancer types nor effective in every patient with animmunogenic cancer type. Cell surface expression of PDL1 ordefects in mismatch repair correlate with therapeutic response insome cancers (23, 24). PDL1 is expressed by solid tumors but isnot expressed in normal epithelial tissues (5), and the CD274 geneencoding PDL1 is amplified in some cases of TNBC (6). Severalother mechanisms leading to increased PDL1 expression havebeen reported (25–27). Hypoxia induces PDL1 expression in tu-mor and immune cells in an HIF-1–dependent manner (28, 29).The failure of many tumors to respond to immune check-

point inhibitors may reflect the multiple immunosuppressivemechanisms employed by cancer cells. Extracellular adenosineis a potent immunosuppressor that accumulates during tumorgrowth (30, 31). Extracellular ATP is converted to AMP by theenzyme CD39, and the subsequent dephosphorylation of AMPto adenosine is catalyzed by the 5′-ectonucleotidase CD73.Adenosine binds to cognate A2A receptors on Teff cells, leadingto anergy or cell death. A2A receptor signaling reduces the cy-totoxic activity of CD8+ T cells and natural killer (NK) cells (32–34). It also increases the number of immunosuppressive Treg cells

and myeloid-derived suppressor cells (MDSCs). A2A receptordeletion or blockade impaired tumor growth and activated tumor-infiltrating lymphocytes (35). CD73 expression is induced by hypoxiain an HIF-dependent manner (30, 36). CD73 expression is increasedin TNBC relative to other breast cancers and is associated withchemotherapy resistance, metastasis, and decreased patient survival(37, 38). Anti-CD73 antibody treatment enhanced the antitumoractivity of anti-PD1 antibody treatment (39).In addition to immune evasion, cancer cells must have the ca-

pacity for self-renewal to form secondary (recurrent or metastatic)tumors. We have previously demonstrated that exposure of breastcancer cells to chemotherapy enriches for cancer stem-like cellsdue to induction of HIF-dependent gene expression (40–42). Inthe present study, we investigated whether exposure to chemo-therapy also induces HIF-dependent changes in CD47, CD73,and PDL1 gene expression that increase the ability of survivingcancer cells to evade innate and adaptive immunity.

ResultsChemotherapy Induces Expression of PDL1, CD47, and CD73 by TNBCCells. SUM159 human TNBC cells were exposed to each of fourdifferent chemotherapy drugs (carboplatin, doxorubicin, gem-citabine, and paclitaxel) for 4 d, at the drug concentration thatinhibited growth by 50%, in a standard 95% air/5% CO2 in-cubator with an ambient O2 concentration of 20%. Reverse

Fig. 1. Chemotherapy induces expression of PDL1, CD73, and CD47. (A–E) SUM159 cells were treated with 50 μM carboplatin, 50 nM doxorubicin, 10 nMgemcitabine, 10 nM paclitaxel, or vehicle for 4 d. RT-qPCR was performed to quantify PDL1 (A), CD47 (B), CD73 (C), HIF-1α (D), and HIF-2α (E) mRNA levelsrelative to 18S rRNA and normalized to vehicle-treated cells (mean ± SEM; n = 3). *P < 0.001 compared with vehicle (by one-way ANOVA with a Bonferroniposttest). (F) Cells were treated with vehicle or carboplatin. After 4 d, the percentage of cells with surface expression of PDL1 (Left), CD47 (Center), or CD73(Right) was determined by flow cytometry (mean ± SEM; n = 3). *P < 0.001 compared with vehicle (by Student’s t test). (G) Cells were treated with vehicle,carboplatin, or paclitaxel as described above. After 4 d, the percentage of cells that were triple-positive for PDL1, CD73, and CD47 was determined (mean ±SEM; n = 3). *P < 0.001 compared with vehicle (by one-way ANOVA with a Bonferroni posttest). All experiments in this figure were performed using cellsexposed to 20% O2 in a standard 95% air/5% CO2 incubator. (H) Analysis of gene expression data from primary human breast cancers. The Pearson correlationtest was performed to compare coexpression of PDL1, CD73, and CD47 mRNA using data from 1,215 breast cancer samples from The Cancer GenomeAtlas (TCGA) database. Pearson’s correlation (r) is shown; P < 0.0001 for all comparisons.

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transcription-quantitative real-time PCR (RT-qPCR) analysisof total RNA isolated from chemotherapy-exposed TNBC cellsrevealed that each of the drugs increased the expression ofPDL1, CD73, CD47, HIF-1α, and HIF-2α mRNA (Fig. 1 A–E).For example, gemcitabine increased PDL1 mRNA by 5.4-fold(Fig. 1A), doxorubicin increased CD73 mRNA by 8.5-fold (Fig. 1C),and carboplatin increased HIF-2α mRNA by 7.9-fold (Fig. 1E).Flow cytometry revealed that, compared with vehicle treatment,carboplatin increased the percentage of cells with surface ex-pression of PDL1, CD47, or CD73 protein by 2.6-, 4.9-, and 6.3-fold, respectively (Fig. 1F). Chemotherapy also increased HIF-1α and HIF-2α protein levels (Fig. S1). We previously reportedthat chemotherapy induces MDR1 expression in an HIF-dependent manner (40), suggesting that there might be differ-ential survival of cells with high HIF activity due to increaseddrug efflux mediated by MDR1. However, the observed in-creases in mRNA and protein expression cannot be due solelyto enhanced chemoresistance, since only 50% of the cells were

killed by chemotherapy, resulting in a maximum enrichment oftwofold due to differential survival.Treatment with carboplatin or paclitaxel increased the per-

centage of triple-positive (PDL1+/CD73+/CD47+) SUM159 cellsby 4.7- and 13-fold, respectively (Fig. 1G). Furthermore, the ob-served frequency of carboplatin-treated cells expressing all threeproteins (1.27%) is 12.7-fold higher than would be expected if theexpression of these proteins was independent of each other(0.10%). Thus, chemotherapy coordinately induces the expressionof PDL1, CD73, and CD47 by surviving TNBC cells.Next, we investigated whether PDL1, CD47, and CD73

mRNAs were expressed in a coordinated manner in 1,215 hu-man breast cancer specimens from The Cancer Genome Atlas(TCGA) database (43, 44) using the Pearson correlation test.PDL1, CD73, and CD47 mRNA levels were significantly correlatedwith each other (P < 0.0001 for all pairwise comparisons) (Fig. 1H).These results are consistent with the coordinate expression of PDL1,CD73, and CD47 in human breast cancer, which implies that thesegenes are subject to similar regulatory mechanisms.

Vehicle Carboplatin Doxorubicin Gemcitabine

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Fig. 2. HIFs mediate chemotherapy-induced PDL1, CD73, and CD47 expression. (A–C) SUM149 cells were treated with vehicle or chemotherapy, either alone or incombination with 2 μM acriflavine, for 4 d. RT-qPCR was performed to quantify PDL1, CD73, and CD47 mRNA levels relative to 18S rRNA and normalized to vehicle-treated cells (mean ± SEM; n = 3). *P < 0.01 compared with vehicle; #P < 0.01 compared with chemotherapy alone (by one-way ANOVA with a Bonferroni posttest).Acr, acriflavine; Carb, carboplatin, Dox, doxorubicin; Gem, gemcitabine; Pac, paclitaxel. (D) Cells were exposed to carboplatin or paclitaxel for 4 d, and flow cytometrywas performed to determine the percentage of triple-positive (PDL1+/CD73+/CD47+) cells (mean ± SEM; n = 3). *P < 0.01 compared with vehicle; #P < 0.01 comparedwith chemotherapy alone (by one-way ANOVA with a Bonferroni posttest). (E) Analysis of gene expression data from primary human breast cancers. The Pearsoncorrelation test was performed to compare expression of HIF-1α, HIF-2α, and HIF signature (HIF Sig) with expression of PDL1, CD47, and CD73 mRNA, using data from1,215 breast cancer samples in the TCGA database. Pearson’s correlation (r) is shown; P < 0.0001 for all comparisons. (F) Immunohistochemistry was performed onbreast cancer biopsies using anti-CD73 and anti–HIF-1α antibodies. (Upper) Representative positive and negative staining (biopsies from patients 2 and 3, respectively)is shown. (Scale bar, 100 μm.) (Lower) Summary of CD73 and HIF-1α expression in 74 human breast cancer biopsies is shown. Neg, negative; Pos, positive.

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Chemotherapy Induces HIF-Dependent Expression of PDL1, CD73, andCD47. To investigate the role of HIFs, we exposed SUM149 TNBCcells to chemotherapy in the absence or presence of the HIF in-hibitor acriflavine, which binds to HIF-1α or HIF-2α and blocks itsheterodimerization with HIF-1β (45). Induction of PDL1, CD47,and CD73 mRNA expression in response to chemotherapy wasblocked by acriflavine (Fig. 2 A–C). Similar results were obtainedusing SUM159 cells (Fig. S2). Acriflavine also blocked the co-ordinate cell surface expression of PDL1, CD73, and CD47 proteinin response to chemotherapy, as determined by detection of triple-positive cells by flow cytometry (Fig. 2D). Remarkably, carboplatinand paclitaxel induced 34-fold and 29-fold increases in the per-centage of PDL1+/CD47+/CD73+ cells (from 0.23% among vehicle-treated cells to 7.9% and 6.8%, respectively, among cells survivingchemotherapy); these effects of chemotherapy were abrogated bycoadministration of acriflavine.We next analyzed TCGA data to determine whether expression

of PDL1, CD47, and CD73 was correlated with expression of HIF-1α

or HIF-2α mRNA, or with an HIF signature, which comprisedHIF-1αmRNA and 13 HIF target-gene mRNAs (PLOD1, VEGFA,LOX, P4HA2, NDRG1, SLC2A1, ERO1L, ADM, LDHA, PGK1,ANGPTL4, SLC2A3, and CA9) in 1,215 breast cancer specimens(43, 44), using the Pearson correlation test. The expressionlevels of PDL1, CD73, and CD47 mRNAs were significantlycorrelated with expression of HIF-1α, HIF-2α, and the HIFsignature (P < 0.0001 in each case; Fig. 2E).Next, we performed immunohistochemistry on adjacent sections

from breast cancer biopsies to analyze CD73 and HIF-1α expres-sion. Sections with >5% stained cells were classified as positive forCD73 or HIF-1α (Fig. 2F, Upper). CD73 was expressed in 36 of 38tumors in which HIF-1α overexpression was detected, comparedwith 24 of 36 tumors in which HIF-1α overexpression was notdetected (Fig. 2F, Lower).

HIFs Directly Activate PDL1, CD73, and CD47 Gene Transcription. Wepreviously demonstrated that HIF-1 directly activated CD47 gene

Fig. 3. HIFs transactivate the PDL1 and CD73 genes. (A) TNBC cell lines were exposed to 20% or 1% O2 for 24 h, and the expression of mRNAs encoding PDL1(Left) and CD73 (Right) was analyzed by RT-qPCR. The expression of each mRNA was quantified relative to 18S rRNA and then normalized to the resultobtained from cells at 20% O2 (mean ± SEM; n = 3). *P < 0.01 versus 20% O2 (by two-way ANOVA with a Bonferroni posttest). (B) Analysis of PDL1 andCD73 mRNA expression in MDA-MB-231 subclones, which expressed an NTC shRNA or shRNA targeting HIF-1α (sh1α), HIF-2α (sh2α), or both HIF-1α and HIF-2α(DKD). Cells were exposed to 20% or 1% O2 for 24 h. Data were normalized to NTC at 20% O2 (mean ± SEM; n = 3). *P < 0.01 versus NTC at 20% O2; **P < 0.01versus NTC at 20% O2;

#P < 0.001 versus NTC at 1% O2 (by two-way ANOVA with a Bonferroni posttest). (C) MDA-MB-231 subclones were exposed to 20% or1% O2 for 72 h, and the percentage of triple-positive cells was determined by flow cytometry (mean ± SEM; n = 3). *P < 0.01 versus NTC at 20% O2;

#P <0.001 versus NTC at 1% O2 (by two-way ANOVA with a Bonferroni posttest). (D and E) MDA-MB-231 cells were exposed to 20% or 1% O2 for 24 h andchromatin immunoprecipitation assays were performed using IgG or antibodies against HIF-1α, HIF-1β, or HIF-2α. Primers flanking the candidate HIF bindingsites were used for qPCR, and results were normalized to lane 1 (mean ± SEM; n = 3). *P < 0.05 versus 20% O2 (by one-way ANOVA with a Bonferroniposttest). The nucleotide sequence (noncoding strand) of HIF binding sites (in red), which are located 5.7 kb 5′ to the transcription start site of the PDL1 gene(D) and within intron 1 of the CD73 gene (E), respectively, are shown. Exons and introns are not drawn to scale.

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transcription when breast cancer cells were exposed to hypoxia (18).Hypoxia-induced expression of CD73 and PDL1 has also beenreported in various cell types (28, 29). To test whether HIFsregulate PDL1 and CD73 expression in human TNBC, we exposedSUM149, SUM159, and MDA-MB-231 cells to 20% or 1% O2 for24 h. Hypoxia induced the expression of PDL1 in two of the threecell lines and CD73 in all three TNBC lines (Fig. 3A).To determine whether HIF-1α or HIF-2α was required for

PDL1 and CD73 expression in TNBC cells, we analyzed MDA-MB-231 subclones, which were stably transfected with an ex-pression vector encoding short hairpin RNA (shRNA) targetingHIF-1α (sh1α), HIF-2α (sh2α), or both HIF-1α and HIF-2α[double knockdown (DKD)], or a nontargeting control (NTC)shRNA, which have been extensively validated and used to in-vestigate the role of HIFs in breast cancer progression (46). Hypoxia-induced expression of PDL1 and CD73 was decreased in the sh1α,sh2α, and DKD subclones (Fig. 3B), indicating a requirement for

both HIF-1α and HIF-2α. These results suggest that intratumoralhypoxia may be responsible for the correlation of CD47, CD73, andPDL1 mRNA expression with the HIF transcriptome in TCGA data(Fig. 2E), which was derived from treatment-naive human breastcancers (44). Hypoxia increased the percentage of NTC cells, but notDKD cells, that were PDL1+/CD47+/CD73+ (Fig. 3C). Thus, hypoxiainduces expression of genes that enable TNBC cells to evade innateand adaptive immunity.We next investigated whether HIF-1 or HIF-2 binds directly to

the PDL1 and CD73 genes. Matches to the HIF binding-site se-quence 5′-(A/G)CGTG-3′ were identified, and HIF binding wasevaluated by chromatin immunoprecipitation assays performedin MDA-MB-231 cells. Hypoxia induced the binding of HIF-1α,HIF-2α, and HIF-1β to sites located in the 5′-flanking region ofPDL1 (Fig. 3D) and in the first intron of CD73 (Fig. 3E). TheHIF binding sites in the CD73 and PDL1 genes identified inMDA-MB-231 cells differ from sites identified in other cell lines

Fig. 4. Exposure of TNBC cells to chemotherapy or hypoxia enables evasion of antitumor T cells. (A) Adenosine levels were measured in CM ofSUM149 and SUM159 cells treated with paclitaxel (5 nM for SUM149 and 10 nM for SUM159) for 4 d. The values were corrected for cell number at the endof the experiment and then normalized to vehicle-treated cells (mean ± SEM; n = 3). *P < 0.05 versus vehicle (by Student’s t test). (B) Adenosine levelswere measured in CM of MDA-MB-231 NTC and DKD subclones exposed to 20% or 1% O2 for 72 h. The values were normalized to NTC at 20% O2 (mean ±SEM; n = 3). *P < 0.05 versus NTC at 20% O2;

#P < 0.05 versus NTC at 1% O2 (by two-way ANOVA with a Bonferroni posttest). (C and D) IFN-γ mRNA levelswere determined (mean ± SEM; n = 3) in activated CD4+ T cells incubated for 24 h with CM (from hypoxic or paclitaxel-treated TNBCs) in the presence orabsence of 4 mM caffeine. *P < 0.05 versus NTC at 20% O2;

#P < 0.05 versus NTC at 1% O2 (by two-way ANOVA with a Bonferroni posttest). (E ) IFN-γmRNAlevels (mean ± SEM; n = 3) were determined in activated CD8+ T cells cultured with CM from MDA-MB-231 subclones exposed to 20% or 1% O2 for 24 h.*P < 0.05 versus NTC at 20% O2;

#P < 0.05 versus NTC at 1% O2 (by two-way ANOVA with a Bonferroni posttest). (F ) IFN-γmRNA levels (mean ± SEM; n = 3)were determined in activated CD8+ T cells incubated for 24 h with CM (from TNBC cells exposed to paclitaxel with or without 2 μM acriflavine for 4 d). *P <0.05 versus vehicle-treated cells; #P < 0.05 versus paclitaxel-treated cells (by one-way ANOVA with a Bonferroni posttest). (G) CD8+ T cells were incubatedwith 4T1 cells (which were pretreated for 4 d with vehicle, acriflavine, paclitaxel, or paclitaxel + acriflavine) in presence or absence of anti-PDL1 blockingantibody. Cell death was assessed by staining with Annexin V and PI. The percentage of CD8+/PD1+ cells that were Annexin V+ and PI+ was quantified byflow cytometry (mean ± SEM; n = 3). *P < 0.05 versus vehicle-treated cells; #P < 0.05 versus paclitaxel-treated cells (by two-way ANOVA with a Bonferroniposttest).

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(28–30). Taken together with our previous study demonstratingHIF-1 binding to the CD47 gene (18), these data indicate that HIFsdirectly activate PDL1, CD47, and CD73 transcription in humanbreast cancer cells, providing a molecular mechanism for coordinateregulation of these genes and their protein products in TNBCs.

Chemotherapy Promotes HIF-Dependent Immune Evasion by TNBCCells. We next investigated whether adenosine production inTNBC cells leads to evasion of adaptive immunity. Cells wereexposed to hypoxia or chemotherapy, culture media were col-lected, and adenosine levels were assayed and normalized to thenumber of cells counted at the end of the experiment. Paclitaxeltreatment of SUM149 and SUM159 cells increased adenosineproduction, particularly in SUM159 cells (Fig. 4A). Hypoxia alsoincreased adenosine production in the NTC subclone of MDA-MB-231, whereas in the DKD subclone, hypoxia-induced adeno-sine production was abrogated (Fig. 4B).

Adenosine inhibits antitumor immunity through binding to A2Areceptors on T cells (47, 48). To test whether exposure of TNBCcells to hypoxia or chemotherapy inhibits antitumor T cells throughadenosine signaling, we cultured activated T cells with conditionedmedia (CM) from TNBC cells that were exposed to hypoxia vs.normoxia or vehicle vs. paclitaxel. We quantified IFN-γ mRNAlevels as a standard measure of T cell activation (49). T cellscultured with CM from TNBC cells that were exposed to hypoxia(Fig. 4C) or paclitaxel (Fig. 4D) exhibited 3.1-fold and 2.3-folddecreased IFN-γ expression, respectively, indicating immunosup-pression. Addition of the adenosine receptor antagonist caffeine(50) blocked the decrease in IFN-γ expression induced by CMfrom TNBC cells exposed to hypoxia (Fig. 4C) or paclitaxel (Fig.4D), demonstrating that the immunosuppression was mediated byadenosine produced in response to hypoxia or chemotherapy.To test whether the decrease in T cell activation induced by

CM from TNBC cells was HIF-dependent, we prepared CM

Fig. 5. Acriflavine blocks paclitaxel-induced enrichment of PDL1+/CD73+/CD47+ TNBC cells and makes the tumor environment less immunosuppressive.(A–C) Mouse 4T1 mammary carcinoma cells were cultured for 4 d in the presence of vehicle, 50 μM carboplatin, 250 nM doxorubicin, 15 nM gemcitabine, or 10 nMpaclitaxel. PDL1, CD47, and CD73 mRNA levels were normalized to vehicle-treated cells (mean ± SEM; n = 3). *P < 0.001 compared with vehicle (by one-way ANOVAwith a Bonferroni posttest). (D) The 4T1 cells were treated with vehicle or paclitaxel in vitro, and flow cytometry was performed to quantify PDL1+/CD73+/CD47+ cells(mean ± SEM; n = 3). *P < 0.001 compared with vehicle (by Student’s t test). (E–G) The 4T1 cells were implanted into the mammary fat pad of female BALB/c mice.When tumor volume reached 200 mm3, mice were treated with: vehicle (saline), acriflavine (4 mg/kg; days 1–13), paclitaxel (10 mg/kg; days 5 and 10), or paclitaxeland acriflavine. Tumors were harvested on day 13, and the percentage of CD45−/PDL1+/CD73+/CD47+ (E), CD8+/CD44+/CD69+ (F), and CD4+/CD25+/FoxP3+ (G) cellswas determined (mean± SEM; n = 4). *P < 0.05 versus vehicle-treatedmice; #P < 0.05 versus paclitaxel-treated mice (by one-way ANOVAwith a Bonferroni posttest).Acr, acriflavine; Pac, paclitaxel. (H–K) The 4T1 cells were implanted into the mammary fat pad of female BALB/c mice. When tumors were palpable, mice weretreated with: vehicle (saline), acriflavine (4 mg/kg; days 1–13), carboplatin (Carb; 20 mg/kg; days 1, 6, and 11), or carboplatin and acriflavine. (H) Tumor volumes weremeasured. Tumors were harvested on day 13, and the percentage of CD8+/CD44+/CD69+ (I), CD4+/CD25+/FoxP3+ (J), and CD11b+/Ly6C+ (K) cells was determined(mean ± SEM; n = 6). *P < 0.05 versus vehicle-treated mice; #P < 0.05 versus paclitaxel-treated mice (by one-way ANOVA with a Bonferroni posttest).

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from MDA-MB-231 NTC and DKD subclones that were ex-posed to 20% or 1% O2 and from SUM159 cells treated withvehicle, acriflavine, paclitaxel, or paclitaxel + acriflavine. IFN-γexpression was inhibited 4.2-fold when activated CD8+ T cellswere incubated with CM from hypoxic NTC cells, but not fromhypoxic DKD cells (Fig. 4E). Incubation of T cells with CM frompaclitaxel-treated SUM159 cells decreased IFN-γ expression by2.6-fold, whereas CM from cells exposed to paclitaxel + acri-flavine did not (Fig. 4F). Thus, HIF-dependent adenosine pro-duction by TNBC cells exposed to hypoxia or chemotherapyinhibits antitumor immunity mediated by both CD4+ (Fig. 4 Cand D) and CD8+ (Fig. 4 E and F) T cells.

Chemotherapy Induces T Cell Apoptosis by Increasing PDL1 Expressionin TNBC Cells. The interaction of PDL1 on cancer cells with PD1 onT cells leads to anergy or cell death. To investigate whetherchemotherapy-induced PDL1 expression by TNBC cells leads toT cell killing, we exposed mouse 4T1 TNBC cells to vehicle, acri-flavine, paclitaxel, or paclitaxel + acriflavine. The 4T1 cells were thencocultured with activated CD8+ T cells, which were subsequentlyanalyzed for apoptosis by propidium iodide (PI) and Annexin Vstaining. Coculture with paclitaxel-treated 4T1 cells led to a 1.8-foldincrease in apoptosis of CD8+/PD1+ T cells, and the effect of pac-litaxel was completely blocked by coadministration of acriflavine orby coculture in the presence of anti-PDL1 antibody (Fig. 4G). Takentogether, the results presented in Fig. 4 demonstrate that exposureof TNBC cells to hypoxia or chemotherapy promotes evasion ofadaptive immunity by inducing HIF-dependent adenosine andPDL1 signaling that mediate increased T cell anergy or apoptosis.

Acriflavine Blocks Chemotherapy-Induced Immune Evasion in a SyngeneicMouse Model. To investigate the role of chemotherapy in immuneevasion in an immunocompetent model of TNBC, we utilized 4T1mouse mammary carcinoma cells, which form primary tumors andmetastases similar to human TNBC after implantation into themammary fat pad of syngeneic BALB/c mice (51, 52). Exposure of

4T1 cells to chemotherapy in vitro induced expression of PDL1,CD73, and CD47 (Fig. 5 A–C), as well as HIF-1α and HIF-2αmRNA (Fig. S3A). Exposure of 4T1 cells to paclitaxel in vitroincreased the percentage of PDL1+/CD73+/CD47+ cells 7.1-fold(Fig. 5D). We verified that acriflavine blocked expression of anHIF target gene (PDK1) in hypoxic 4T1 cells in a dose-dependentmanner but had no effect on the expression of RPL13A, which isnot an HIF target gene (Fig. S3B).To investigate whether paclitaxel elicited similar effects in vivo,

we implanted 4T1 cells in the mammary fat pad of female BALB/cmice. When the tumors reached a volume of 200 mm3 (designatedday 1), mice were randomized to receive i.p. injections of saline,acriflavine (4 mg/kg on days 1–13), paclitaxel (10 mg/kg on days5 and 10), or both acriflavine and paclitaxel. Tumors were har-vested on day 13. There was no effect of paclitaxel, acriflavine, orpaclitaxel + acriflavine on tumor growth or body weights of themice (Fig. S4). Freshly harvested tumors were dissociated intosingle-cell suspensions and subjected to flow cytometry. Comparedwith vehicle, paclitaxel increased CD45−/PDL1+/CD73+/CD47+

tumor cells by 3.3-fold, and this effect was blocked by acriflavine(Fig. 5E). Thus, TNBC cells that survive cytotoxic chemotherapymanifest increased expression of genes encoding immunosup-pressive proteins, whereas coadministration of an HIF inhibitorblocks chemotherapy-induced expression of gene products thatmediate immune evasion.Next, we analyzed immune cells within the tumor. Paclitaxel

treatment decreased intratumoral CD8+/CD44+/CD69+ Teff cellsby 3.7-fold, whereas acriflavine coadministration restored Teffcells to levels that were not significantly different from tumors insaline-treated mice (Fig. 5F). Paclitaxel treatment significantlyincreased CD4+/CD25+/FoxP3+ Treg cells by 2.2-fold, and thiseffect was completely blocked by acriflavine (Fig. 5G).We next investigated whether similar effects would be ob-

served if tumor-bearing mice were treated as soon as tumorsbecame palpable, rather than after extensive tumor growth, andwere treated with carboplatin, instead of paclitaxel (Fig. 5H and

Fig. 6. Chemotherapy promotes immune evasion phenotype in surviving TNBC cells. Exposure of TNBC cells to cytotoxic chemotherapy (or hypoxia)induces expression of HIF-1α and HIF-2α, leading to the HIF-mediated expression of PDL1, CD73, and CD47, which promote suppression of innate anti-tumor immunity mediated by macrophages, dendritic cells (DCs), and MDSCs, and suppression of adaptive antitumor immunity mediated by T cells. Ado,adenosine; IDO, indoleamine-2,3-dioxygenase.

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Fig. S5 A and B). As in the case of paclitaxel, we observed thatcompared with vehicle, carboplatin treatment significantly de-creased the percentage of intratumoral CD8+/CD44+/CD69+ Teffcells (Fig. 5I) and increased the percentage of intratumoral CD4+/CD25+/Foxp3+ Treg cells (Fig. 5J). The intratumoral Teff/Treg cellratio fell from 5.92 in vehicle-treated mice to 0.31 in carboplatin-treated mice, a 19-fold decrease. In contrast, coadministration ofacriflavine abrogated the carboplatin-induced Teff cell decreaseand Treg cell increase in the tumor, restoring a 3.8-fold excess ofTeff cells over Treg cells. In addition, carboplatin induced a 1.8-foldincrease in intratumoral CD11b+/Ly6C+ MDSCs, which was alsoabrogated by coadministration of acriflavine (Fig. 5K).In contrast to the effects on T cells and MDSCs, carboplatin

treatment had no effect on the percentage of intratumoral CD3−/NK1.1+ NK cells, CD3−/CD19+ B cells, or CD11b+F4/80+

tumor-associated macrophages (Fig. S5 C–E). Taken together,the results presented in Fig. 5 indicate that cytotoxic chemotherapyinduces HIF-dependent enrichment of PDL1+/CD73+/CD47+

TNBC cells in vivo and makes the tumor microenvironmentmarkedly less immunogenic by specifically increasing both Tregcells and MDSCs, as well as decreasing Teff cells. Coadministra-tion of acriflavine is sufficient to block all of the observedcountertherapeutic effects of cytotoxic chemotherapy on antitu-mor innate and adaptive immunity.

DiscussionRecent studies have demonstrated the role of the immune sys-tem in promoting cancer cell death in response to chemotherapy(3). In the present study, however, we have focused on the cancercells that survive chemotherapy. Prior studies reported that hyp-oxia induces CD47, CD73, and PDL1 gene expression in anHIF-dependent manner (18, 28–30, 32). We previously dem-onstrated that treatment of tumor-bearing mice with HIF in-hibitor blocked recruitment of MDSCs to orthotopic TNBCs(50) and that knockdown of HIFs or CD47 led to increasedphagocytosis of breast cancer cells by macrophages (18). In thepresent study, we have demonstrated effects of chemotherapy onimmune cells that are due to HIF-mediated gene expression inTNBC cells. Our results indicate that for multiple chemotherapyagents and multiple TNBC cell lines, chemotherapy induces HIF-dependent, coordinate transcriptional activation of PDL1, CD47,and CD73 expression. Cell surface expression of PDL1 and/orCD73 enables TNBC cells to induce anergy or apoptosis of Teffcells with a concomitant increase in Treg cells in the tumor mi-croenvironment, thereby impairing adaptive antitumor immunity.One limitation of in vitro exposure of cancer cells to chemo-

therapy is that it excludes the role of different immune cell typesin either enhancing or inhibiting chemotherapy-induced cancercell death (53). Another caveat is that serum in tissue culturemedia contains high levels of adenosine deaminase, which mayresult in an underestimation of cellular adenosine production invitro. However, despite these caveats, there was a striking con-cordance of results from in vitro and in vivo studies demon-strating that chemotherapy induces CD47, CD73, and PDL1expression and an immunosuppressive tumor microenvironment inan HIF-dependent manner.Although we demonstrated surprisingly strong inhibitory ef-

fects of chemotherapy on antitumor immunity in vivo, a caveatof these studies is that intratumoral immune cell populationswere analyzed using a single mouse strain, cancer cell line,chemotherapy dose, and time point. Thus, BALB/c mice weretreated with 10 mg/kg of paclitaxel every 5 d, and 4T1 ortho-topic tumors were analyzed 3 d after the last dose of paclitaxel,revealing a markedly increased percentage of Treg cells com-pared with tumors from vehicle-treated mice. In a previouslypublished study, C57BL/6 mice bearing 3LL Lewis lung tumorxenografts were treated with paclitaxel at a dose of 10 mg/kgeach day for 3 d and analyzed 1 d later, revealing a significant

decrease in Treg cells (54). Further studies are required to in-vestigate which of the many differences between these twostudies were responsible for the divergent outcomes. Weobtained remarkably similar results when we repeated the ex-periment using a different chemotherapy agent (carboplatin)and different tumor size (first palpable rather than 200 mm3).In both experiments, chemotherapy-induced increases in Tregcells were accompanied by reciprocal decreases in Teff cells,and all of the effects were blocked by coadministration of theHIF inhibitor acriflavine. In addition to inhibiting CD73 andPDL1 expression in TNBC cells, acriflavine administration mayaffect the Treg/Teff cell ratio by inhibiting HIF-dependent ex-pression of FOXP3 (14), which is a critical determinant of Tregcell differentiation.The coordinate induction of PDL1, CD47, and CD73 in re-

sponse to chemotherapy endows TNBC cells with the ability toevade both innate and adaptive immune systems (Fig. 6). Ourresults provide a rationale for combining chemotherapy andimmunotherapy (anti-PD1 or anti-PDL1) to improve the out-come of patients with TNBC. Our results also offer a potentialexplanation for the limited response of cancer patients to im-mune checkpoint inhibitors (55). If multiple mechanisms of im-mune evasion are coordinately regulated by HIFs, then targeting anysingle pathway (e.g., by anti-PD1, anti-PDL1, or adenosine receptorantagonist therapy) may be insufficient to restore antitumor immu-nity in a tumor with high HIF activity. An alternative approach is tocombine cytotoxic chemotherapy with immune checkpoint inhibi-tors. However, an improvement in the therapeutic response ofmice bearing Brca1−/− TNBC tumors (which have a high mutationload due to defective DNA repair) to cisplatin required combinedtreatment with both anti-CTLA4 and anti-PD1 antibodies (56),which is a treatment regimen that has been associated with con-siderable toxicity (57).Prior studies delineated multiple HIF-dependent pathways

leading to the induction of tumor-initiating cells [also known asthe breast cancer stem cell-like (BCSC) phenotype] in responseto chemotherapy (40–42). CD47 and CD73 have been reportedto promote the BCSC phenotype through mechanisms that arenot understood (18, 58). Given that patients with TNBC in re-mission after chemotherapy may harbor millions of cancer cellsor more, the finding that 0.7% of TNBC cells that survivedpaclitaxel therapy in vivo expressed proteins that mediate bothimmune evasion and tumor initiation provides a molecular mech-anism for TNBC recurrence, metastasis, and patient mortality.Coadministration of HIF inhibitor blocks chemotherapy-inducedexpression of genes encoding the BCSC phenotype (40–42) andimmune evasion as demonstrated in the present study. Thus, co-administration of HIF inhibitors with chemotherapy might im-prove the survival of patients who have TNBC.Our results demonstrate that HIF inhibitors block the counter-

therapeutic effect of paclitaxel and other cytotoxic chemotherapyagents in promoting immune evasion by TNBC cells. HIF inhibi-tors might serve as broad-spectrum inducers of antitumor immu-nity, even in tumors such as 4T1 that express a very limited numberof mutant epitopes (59). Further studies are required to determinewhether, by blocking the expression of multiple proteins that me-diate evasion of the adaptive and innate immune systems, HIFinhibitors might also improve responses to immune checkpointinhibitors, adoptive cell transfer, or drugs targeting the adenosinesignaling pathway.

Materials and MethodsCell Lines. MDA-MB-231 cells were maintained in high-glucose (4.5 mg/mL)Dulbecco’s modified Eagle medium (DMEM) with 10% (vol/vol) FBS and 1%penicillin/streptomycin. SUM159 and SUM149 cells were maintained inDMEM/F12 (50:50) with 10% FBS, hydrocortisone, insulin, and 1% penicillin/streptomycin. MDA-MB-231 shRNA subclones were cultured in the presenceof 0.5 μg/mL puromycin. The 4T1 cells were maintained in RPMI-1640 with

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10% FBS and 1% penicillin/streptomycin. Cell line identity and absence ofmycoplasma infection were validated by PCR-based assays. Cells weremaintained at 37 °C in a 5% CO2/95% air incubator (20% O2). Hypoxic cellswere maintained at 37 °C in a modular incubator chamber (Billups–Rothenberg)flushed with a gas mixture containing 1% O2, 5% CO2, and 94% N2. Pacli-taxel, doxorubicin, acriflavine, and gemcitabine were obtained from Sigma–Aldrich and dissolved in DMSO at 1,000× relative to final concentration intissue culture medium. Caffeine was obtained from Sigma–Aldrich and dis-solved in deionized water.

RT-qPCR. Total RNA was extracted from cells using TRIzol (Invitrogen) andtreated with DNase I (Ambion). One microgram of total RNA was used forfirst-strand DNA synthesis with the iScript cDNA Synthesis system (BioRad).qPCRwas performed using SYBR Green qPCRMaster Mix (BioRad). For eachprimer pair (nucleotide sequences are shown in Table S1), the annealingtemperature was optimized by gradient PCR. The expression of eachtarget mRNA relative to 18S rRNA was calculated based on the thresholdcycle (Ct) as 2−Δ(ΔCt), where ΔCt = Cttarget − Ct18S and Δ(ΔCt) = ΔCttest −ΔCtcontrol.

Immunoblot Assays.Whole-cell lysates were prepared in radioimmunoprecipitationassay lysis buffer. Blots were probed with antibodies against HIF-1α andHIF-2α (Table S2). HRP-conjugated anti-rabbit (Roche) and anti-mouse(Santa Cruz Biotechnology) secondary antibodies were used. The chemi-luminescent signal was detected using ECL Plus (GE Healthcare). Blots werestripped and reprobed with antibody against actin (Santa Cruz Bio-technology) to confirm equal protein loading.

Flow Cytometry. Cultured cells were trypsinized, whereas tumor tissues wereminced, digested with 1 mg/mL type 1 collagenase (Sigma) at 37 °C for 30 min,and filtered through 70-μm cell strainers. Cells were incubatedwith Fc Block (BDPharmingen). PDL1+/CD47+/CD73+ cells were identified by staining with fluo-rescein isothiocyanate (FITC)-conjugated anti-PDL1, phycoerythrin-conjugatedanti-CD47, and allophycocyanin (APC)-conjugated anti-CD73 antibodies,and quantified by flow cytometry. Activated Teff cells were identified bystaining with APC-conjugated anti-CD8, FITC-conjugated anti-CD69, andAF405-conjugated anti-CD44 antibodies, and subjected to flow cytometry.Treg cells were identified by staining with APC-conjugated anti-CD4, FITC-conjugated anti-CD25, and AF405-conjugated anti-FoxP3 antibodies, andquantified by flow cytometry. All fluorescent antibodies were from NovusBiologicals (Table S2). Unstained control and single-stained cells were pre-pared in every experiment for gating. Dead cells were gated out by side-scatter and forward-scatter analysis.

Chromatin Immunoprecipitation. MDA-MB-231 cells were incubated at 20%or 1% O2 for 24 h, cross-linked in 3.7% formaldehyde for 15 min,quenched in 0.125 M glycine for 5 min, and lysed with SDS lysis buffer.Chromatin was sheared by sonication, and lysates were precleared withsalmon sperm DNA/protein A agarose slurry (Millipore) for 1 h and in-cubated with IgG or antibody against HIF-1α, HIF-1β, or HIF-2α (Table S2)in the presence of protein A-agarose beads overnight. After washes ofthe agarose beads with low-salt, high-salt, and LiCl buffer, DNA waseluted in 1% SDS with 0.1 M NaHCO3, and cross-links were reversed byaddition of 0.2 M NaCl. DNA was purified by phenol-chloroform ex-traction and ethanol precipitation, and analyzed by qPCR (Table S3).

Adenosine Measurements. Cells were counted, plated, and exposed tohypoxia for 3 d or to chemotherapy for 4 d. CM were collected andcentrifuged to pellet cell debris. Adenosine levels were determinedbased on a standard curve according to the manufacturer’s instructions(BioVision).

T Cell Isolation. Naive CD4+ T cells (CD4+/CD25−/CD62L+) or CD8+ T cells wereisolated from wild-type C57BL/6 mice by magnetic immunoseparation (cat-alog nos. 130-104-453 and 130-104-075, respectively; Miltenyi) and activatedwith anti-CD3 and anti-CD28 (Bio Legend) antibodies in a 24-well plate (1 μgand 4 μg per well, respectively) overnight.

IFN-γ mRNA Expression. CM from MDA-MB-231 cells exposed to hypoxia for3 d and SUM159 cells treated with chemotherapy for 4 d were collected. Fivehundred microliters of CM was lyophilized. CD4+ or CD8+ T cells were acti-vated and then incubated for 24 h with the lyophilized CM dissolved in freshmedia with or without 4 mM caffeine. At the end of the experiment, RNAwas extracted and RT-qPCR was performed.

Coculture Assays. SUM159 cells were treated with vehicle, paclitaxel, acri-flavine, or paclitaxel + acriflavine for 4 d. The surviving cells were counted,and an equal number of live cells were plated for each condition. The nextday, activated CD8+ T cells were plated in the wells containing the cancercells and cocultured for 48 h, and cell death was quantified by stainingwith FITC-conjugated anti-Annexin V antibody and PI, along with AF405-conjugated anti-CD8 and APC-conjugated anti-PD1 antibodies. Unstainedcontrol and single-stained cells were prepared in every experiment forgating. The percentage of CD8+/PD1+/AnnexinV+/PI+ cells was determinedby flow cytometry.

Mouse Studies. Animal protocols were in accordance with the National Insti-tutes of Health Guide for the Care and Use of Laboratory Animals (60)and were approved by The Johns Hopkins University Animal Care andUse Committee. Female 5- to 7-wk-old BALB/c mice (Charles River Lab-oratories) were studied. Carboplatin, paclitaxel, and saline for injectionwere obtained from the research pharmacy of The Johns Hopkins Hos-pital. Cultured 4T1 cells were harvested by trypsinization, rinsed withPBS, and resuspended at 5 × 105 cells per milliliter in a 1:1 solution ofPBS/Matrigel. The 4T1 cells were injected into the mammary fat pad.Primary tumors were measured in three dimensions (a, b, and c), andvolume (V) was calculated as V = abc × 0.52.

Immunohistochemistry. We utilized a tissue microarray of invasive breastcarcinomas that were represented by triplicate punched core samples(0.6 mm in diameter) selected from paraffin tissue blocks of surgicallyresected primary cancers. An exemption for the use of deidentified humantumor tissue was approved by the Johns Hopkins Institutional ReviewBoard. The following antibodies and staining methods were used: forCD73, Novus antibody NBP1-85740 was incubated at a 1:1,000 dilution for1 h at room temperature; for HIF-1α, Novus antibody NB100-105 was in-cubated at a 1:100 dilution at 4 °C overnight after target antigen retrieval incitrate buffer (pH 6) in a pressure cooker for 3 min on high.

Statistical Analysis. Data are expressed as mean ± SEM. Differences betweentwo groups or multiple groups were analyzed by a Student’s t test or one-way ANOVA followed by a Bonferroni posttest, respectively. The Pearsoncorrelation test was performed to compare mRNA expression data from1,215 TCGA breast cancer samples (44). The Graphpad Prism (Version 6.0)software package was used.

ACKNOWLEDGMENTS. We thank Karen Padgett of Novus Biologicals forproviding IgG and antibodies against PDL1, CD73, CD47, CD45, CD4, CD8,CD44, CD69, and FOXP3. This work was supported by the EmersonCollective Cancer Research Fund, American Cancer Society, and CindyRosencrans Fund for Triple-Negative Breast Cancer. G.L.S. is an AmericanCancer Society Research Professor and the C. Michael Armstrong Pro-fessor at The Johns Hopkins University School of Medicine.

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