Breast Cancer Stem Cell–Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands

Patient-derived triple-negative BCSCs are shown to be efficiently recognized and killed by γδ T cells, but xenografted BCSCs evade immune recognition by γδ T cells by various mechanisms, paving the way for novel combinatorial immunotherapies for TNBC.


Introduction
In breast cancer, which was the most commonly diagnosed cancer worldwide in 2020, breast cancer stem cells (BCSC) play a key role in metastasis formation, tumor recurrence, and mortality of patients (1,2). Specifically targeting BCSCs is a promising avenue for cancer therapy, yet it faces multiple challenges mainly due to BCSC-intrinsic cell heterogeneity and drug resistance (3). BCSCfocused therapies are of even greater importance in the fight against triple-negative breast cancer (TNBC), the most aggressive and lethal breast cancer subtype, for which no targeted treatment options are currently available (4).
Immunotherapies involving the transfer of autologous ex vivo engineered ab T cells back into patients have recently been employed for treating a variety of cancers. However, the efficacy of these treatments relies on the presence of tumor-specific antigens presented on MHC I molecules. The loss of MHC I expression and the concomitant lack of peptide presentation by cancer cells can undermine conventional ab T-cell target recognition, and thus lead to tumor immune escape, which has been widely observed in cancers, including TNBC (5). To overcome this limitation, novel immunotherapies using unconventional non-MHC-restricted lymphocytes such as gd T cells are currently being investigated. gd T cells react to stress-induced proteins or phosphoantigens (pAgs) that accumulate in tumor cells due to their deregulated metabolism (6,7).
Two subpopulations of gd T cells are currently in focus for therapeutic applications: Vg9Vd2 T-cell receptor (TCR)-expressing gd T cells (Vd2 þ T cells) and Vd1 þ gd T cells (Vd1 þ T cells). Vd2 þ T cells represent the predominant gd T-cell subset in the human blood and specifically recognize pAgs (8,9). In contrast, Vd1 þ T cells represent a minor population in peripheral human blood and mainly locate to epithelial tissues. Vd1 þ T cells react to several lipid and protein antigens (10). Both of these gd T-cell subsets have been described to kill a variety of cancer cell lines upon activation of their specific TCRs, innate receptors like NKG2D, or by engaging the "death receptors" Fas or TRAIL on tumor cells (11)(12)(13). We investigated here the potential of these two gd T-cell subsets to target BCSCs.
Reactivity of gd T cells against cancer stem cells of several cancer entities has been described previously (14,15). Only two recent studies addressed the reactivity of gd T cells against BCSCs (16,17). While Chen and colleagues did not observe a difference in gd T cell-mediated cytotoxicity between BCSCs and their non-stem cell counterparts, Dutta and colleagues reported that BCSCs were less susceptible to being killed by gd T cells. To reconcile these discrepancies and to shed light onto the potential of gd T cells to target BCSCs, studies better reflecting the clinical reality are urgently needed.
The potential success of gd T-cell immunotherapy against solid tumors also relies on the efficient localization of these cells into the tumor tissue because T cells require direct cell-cell contact to exert their cytotoxicity. gd T cells need to extravasate from the blood stream into the tissue and migrate in the stromal tumor compartment. It has been shown for conventional ab T cells that the extracellular matrix (ECM), the major noncellular fraction of the tumor microenvironment, negatively affects the migration and infiltration of ab T cells in non-small cell lung cancer and ovarian cancer (18,19). High stromal content, as well as low numbers of infiltrating T cells, also have been associated with poor clinical outcome in breast cancer (20). Therefore, increasing the infiltration of T cells into tumors is a major goal in the development of immunotherapies. In line with this, exogenous expression of the ECM-modifying enzyme heparanase can increase the tumor infiltration of chimeric-antigen receptor (CAR)-expressing ab T cells, promoting tumor rejection in melanoma and neuroblastoma xenograft models (21). Whether the ECM-rich stromal compartment of tumors also hampers gd T-cell migration, infiltration, and tumor rejection has not yet been investigated.
We sought here to use a model aiming to reflect the situation in human patients with TNBC to test the effect of gd T cell-based therapy.

gd T-cell expansion
Primary gd T-cell expansion was performed as described previously (23). Briefly, peripheral blood mononuclear cells (PBMC) were purified from the blood of healthy donors via density gradient centrifugation (Pancoll human, Pan Biotech, P04-601000). Buffy coats used as blood source were purchased from the blood bank of the University Medical Centre Freiburg (approval of the University Freiburg Ethics Committee: 147/15 and 21-1010). PBMCs were resuspended at a concentration of 1 Â 10 6 cells/mL in gd T-cell medium: RPMI1640 medium (Life Technologies, 11554516) supplemented with 10% FBS, 100 mg/mL penicillin, 100 mg/mL streptomycin, 10 mmol/L HEPES, 10 mmol/L sodium pyruvate, and 1Â MEM nonessential amino acids (Pan Biotech, P08-32100). Expansion was induced with 1 mg/mL concanavalin A (Sigma-Aldrich, C5275), 10 ng/mL IL2 (Peprotech, 200-02), and 10 ng/mL IL4 (Peprotech, 200-04). Cells were adjusted every 3 to 4 days to 1 Â 10 6 cells/mL with gd T-cell medium and cytokines. The cells were cultured at 37 C in a humidified atmosphere of 5% CO 2 . At days 10 to 14 after expansion started, gd T cells were separated from ab T cells via negative selection (MACS TCRg/d þ T Cell Isolation Kit, Miltenyi Biotec, 130-092-892). Cultures with a purity of ≥ 95% were used for experiments from day 14 on.
Lentiviral constructs, generation of lentiviral particles, and transduction of gd T cells MMP14 cDNA or the cDNA of the catalytically inactive form of MMP14 (MMP14 E240A ; both kind gifts from Pilar Gonzalo, CNIC, Spain and Joaquin Teixid o, CIB, Spain) were cloned into the lentiviral backbone pLVX-CMV-IRES-zsGreen1 (Takara/Clontech #632187) by Gibson assembly. The CMV promoter was exchanged with a short EF-1a promoter sequence (GATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACAT-CGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAA-TTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGG-GAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGG-TGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAAC-GTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTC-GTGACGCG). The integrity of each plasmid was verified by restriction enzyme digestion and Sanger sequencing.
For the generation of lentiviral particles, 1 Â 10 7 HEK 293T cells were plated on 15 cm dishes and cultured at 37 C and 7.5% CO 2 . After 24 hours, the medium was exchanged and HEK293T cells were transfected with the indicated constructs and the packaging plasmids pCMV-dR8.74 and pMD2-vsvG (both kind gifts from Didier Trono, EPFL, Switzerland) using polyethylenimine transfection (polysciences, 24765). After 24 and 48 hours, the viral particle-containing supernatant was harvested, pooled, filtered, and concentrated using density centrifugation [10% sucrose w/v in PBS/0.5 mmol/L Ethylenediaminetetraacetic acid (EDTA)] for 4 hours at 10,000 Â g and 6 C. The supernatant was discarded, and the viral particles were resuspended in PBS using 1/400th of the harvested volume and stored at À80 C.
gd T cells were lentivirally transduced with a multiplicity of infection (MOI) of 2-5 as indicated for the individual experiments. Transduced gd T cells were checked for zsGreen1 and matrix metalloprotease 14 (MMP14) expression by flow cytometry after 2 to 3 days after transduction.

Flow cytometry analysis
To stain cell surface proteins, cells were washed once with flow cytometry buffer (PBS supplemented with 2% FBS) and incubated in the diluted antibody solution for 15 minutes at 4 C. In the case of fluorophore-labeled antibodies, cells were washed once with flow cytometry buffer and analyzed on a Gallios flow cytometer (Beckman Coulter). The flow cytometry results were analyzed using FlowJo v10.8 Software (BD Life Sciences). Unlabeled primary antibodies were visualized using fluorescently labeled secondary antibodies. After washing away the primary antibody, cells were incubated in the diluted secondary antibody solution for 15 minutes at 4 C. Finally, cells were washed once as described above and analyzed on a Gallios flow cytometer.
Viability of gd T cells was assessed using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, 556547).

Cytotoxicity and degranulation assay
Bioluminescence-based cytotoxicity assays were performed as described previously (24). Briefly, 1 Â 10 4 luciferase-expressing target cells were plated in a white 96-well flat-bottom plate. Effector cells were added to the target cells at the desired effector to target cell ratios. A total of 37.5 mg/mL D-Luciferin Firefly (Biosynth, FL08608) was added to the samples, which were then incubated for the indicated time at 37 C and measured using a luminometer (Tecan infinity M200 Pro). Bioluminescence was measured as relative light units (RLU). RLU signals from target cells alone served as spontaneous death controls. Maximum killing RLUs were determined using target cells lysed with 1% Triton X-100. The specific lysis was calculated with the following formula: %specific lysis ¼ 100x ðspontaneous death RLU À testRLUÞ ðspontaneous death RLU À maximum killing RLUÞ 51 Cr-release assays were performed to assess cytotoxicity in experiments including xenograft-derived tumor cells. Target cells (tumor cells) were loaded with 51 Cr (PerkinElmer, NEZ030005MC) for 1 hour at 37 C. After washing the cells three times with medium, cells were resuspended in gd T-cell medium and 1 Â 10 4 cells were plated in a 96well U bottom plate. Effector cells (gd T cells) were added to the target cells at the desired effector to target cell ratios. Samples were incubated for the indicated time at 37 C. Supernatants were transferred to a solid scintillator-coated 96-well plate (LumaPlate, Perkin Elmer) and then measured using a microplate scintillation g-ray counter (TopCount, Perkin Elmer). Target cells without effector cells served as spontaneous 51 Cr release controls. Maximum 51 Cr release was determined using target cells lysed with 1:20 centrimide. The specific lysis was calculated with the following formula: % specific lysis ¼ 100x ðtest Cr51 release À spontaneous Cr51 releaseÞ ðmaximum Cr51 release À spontaneous Cr51 releaseÞ For blocking experiments, effector or targets cells were preincubated with 20 mg/mL of the indicated blocking antibodies at 37 C for 1 hour. IgG isotype antibodies were used as experimental controls. Cells were then used for cytotoxicity experiments in the presence of 10 mg/mL of the blocking antibodies detailed above (see Antibodies and chemicals).
The blocking agent mevastatin was preincubated with the target cells at a concentration of 25 mmol/L for 2 hours at 37 C. The cytotoxicity assay was conducted in the presence of 25 mmol/L mevastatin. Experiments with zoledronate were performed depending on the target cells. BCSC culture cells were preincubated with 10 mmol/L zoledronate over night at 37 C and washed before the cytotoxicity assay. In contrast, for experiments including xenograftderived tumor cells, all target cells were preincubated with 10 mmol/L zoledronate for 2 hours and the cytotoxicity assay was performed in the presence of 10 mmol/L zoledronate.
To analyze gd T-cell degranulation, BCSCs or xenograft-derived tumor cells were labeled with 20 mmol/L cell proliferation dye eFluor450 (Thermo Fisher Scientific). A total of 1 Â 10 5 tumor cells and 1 Â 10 5 gd T cells were cocultured for 3 hours at 37 C in the presence of 1 mL anti-CD107a-PE (BD Biosciences). Medium or stimulation with anti-CD3 and anti-CD28 (both 1 mg/mL; platebound) served as negative and positive controls, respectively. Cells were harvested and analyzed by flow cytometry.

IFNg ELISA
A total of 1 Â 10 5 gd T cells were cocultured with tumor cells for 24 hours at a ratio of 1:1. Target cells were preincubated with 20 mmol/L zoledronate for 2 hours or with 2 Â 10 3 U or 2 Â 10 4 U IFNa 2B for 1 hour at 37 C before coculturing them with gd T cells. The assay was conducted with a final concentration of 10 mmol/L zoledronate or 1 Â 10 3 U or 1 Â 10 4 U IFNa 2B. The culture supernatant was then analyzed for secreted IFNg using an IFNg ELISA kit (Thermo Fisher Scientific, EHIFNG) according to the manufacturer's instructions.
Xenograft tumor model and gd T-cell treatment NOD SCID mice (NOD.CB17-Prkdcscid/Rj, Janvier Labs) and Rag2-g-mice (Rag2tm1.1Flv IL-2rgtm1.1Flv, Jackson Laboratory) were housed at the Center for Experimental Models and Transgenic Service, Freiburg, under specific pathogen-free conditions using individually ventilated cages. Mouse handling and experiments were performed in accordance with German Animal Welfare regulations and approved by the Regierungspr€ asidium Freiburg (animal protocols G17/137 and G19/168).
The orthotopic transplant was performed as described previously (22). A total of 5 Â 10 5 BCSCs were mixed with 1 Â 10 6 irradiated HDF in a 1:1 mixture of MSC medium and Matrigel (growth factor reduced, Corning) and transplanted into each fat pad of the two #4 mammary glands of female NOD SCID mice. Mice were anesthetized during the procedure using an isoflurane inhalator. Before treatment started, mice were randomized into three groups: vehicle (PBS), gd T cell, gd T cell MMP14. Treatment was initiated for each mouse individually at a tumor volume of at least 4 mm 3 as indicated for each experiment. A total of 5 Â 10 6 gd T cells (culture purity ≥95%; <5% Vd1 þ ) were intravenously injected three times per week for the indicated time period. In addition, mice received 0.6 Â 10 6 IU of IL2 (Proleukin, Novartis) in incomplete Freund's adjuvant subcutaneously in the abdomen to support gd T-cell survival in vivo. Checkpoint inhibition using the a-PD-1 antibody Nivolumab (Opdivo, Bristol Myers Squibb) and a corresponding a-human IgG4k isotype control (H€ olzel Diagnostika Handels GmbH, HG4K-25) was performed by additional biweekly intraperitoneal injections of 200 mg of the respective antibody. Tumor sizes were defined by caliper measurement. Tumor volumes were calculated using the formula V ¼ 4/3 Â p Â r 3 .

Preparation of tumor single-cell suspensions
Tumors from BCSC5 orthotopic xenografts (see Xenograft tumor model) were cut into small pieces using a razor blade and digested with 1 mg/mL collagenase IV (Sigma-Aldrich, C5138) and 0.1 mg/mL DNAse I (Roche, 37770400) at 37 C for 45 minutes. The digested tissue was filtered through 70 and 40 mm filters. To remove remaining cell debris, the cell suspension was cleared via centrifugation through a FBS layer. Erythrocytes were removed using Ammonium-Chloride-Potassium (ACK) lysis and recovered cells were used for experiments when the proportion of human EpCAM-positive (EpCAM þ ) cells was > 95%. For flow cytometric analysis, EpCAM þ cells were gated.

Preparation of murine blood and liver samples
Leukocytes were isolated from blood samples by repeated erythrocyte lysis steps using ACK lysis buffer. When only minimal residual erythrocytes were left in the sample, cells were used for flow cytometric analysis. To analyze liver-derived lymphocytes, liver tissue was cut into small pieces using scissors and further dissociated through a 70 mm filter. The cell suspension was centrifuged at 60 Â g for 1 minute at room temperature without break and the resulting supernatant was centrifuged at 850 Â g for 8 minutes at room temperature. The pellet was then resuspended in 10 mL 37.5% Percoll (Sigma-Aldrich, P4937) in PBS and 100 U/mL heparin (Sigma-Aldrich, H3149) and centrifuged at 850 Â g for 20 minutes at room temperature without break. Erythrocytes were removed from the pellet using ACK lysis and recovered cells were used for flow cytometry.

Preparation of viable BCSC5 xenograft tumor slices and confocal imaging of gd T-cell migration
Tumor slices were prepared as described previously (18). Briefly, BCSC5 xenograft-derived tumors were cut into small pieces using a razor blade. Tumor pieces were embedded in a 5% low-gellingtemperature agarose (Sigma-Aldrich, A9045) solution (w/v in PBS). After agarose solidification at 4 C, agarose blocks were fixed on the specimen disk of a vibratome using nontoxic tissue adhesive (3M Vetbond, 1469c). The embedded tissue was cut into 350-mm-thick slices in ice-cold PBS. Tumor slices were transferred onto 30 mm organotypic culture insert (Merck), which had been placed in the wells of a 6-well plate filled with 1.1 mL phenol red-free RPMI medium (Thermo Fisher Scientific, 11835030) supplemented with 10% FBS, 100 mg/mL penicillin, and 100 mg/mL streptomycin (slice assay medium).
For fluorescent labeling of the tumor tissue and plating of gd T cells, prewet stainless steel flat washers were placed onto the agarose surrounding each tumor slice and the slices were incubated at 37 C for 10 minutes. Then, tumor slices were stained with a-EpCAM-BV421 (10 mg/mL) and anti-fibronectin (3.5 mg/mL) for 15 minutes at 37 C and subsequently washed with slice assay medium. A total of 1 Â 10 6 gd T cells were labeled with 0.5 mmol/L CellTracker Green CMFDA Dye (Thermo Fisher Scientific) according to the manufacturer's instruction and mixed with an anti-rabbit-AlexaFluor564 (10 mg/mL). The solution was added on the tumor slices and incubated for 30 minutes at 37 C. Subsequently slices were washed and incubated at 37 C for 10 minutes until imaging.
Image acquisition was performed at 37 C in slice assay medium with a LSM 880 inverted laser scanning confocal microscope (Zeiss) equipped with a 25Â objective (LD LCI Plan-Apochromat, NA 0.8, WD 0.57 mm, water immersion) using the Fast Airyscan mode. BV421, CMFDA, and AlexaFluor546 were excited with a 405, 488, or 561 nm laser, respectively. Nine optical planes spanning a total depth of 63 mm in the Z dimension were captured every 30 seconds for 20 to 45 minutes.
Airyscan data were first processed and stitched using the Zeiss Zen Black edition 3.0 SR. Then, ECM regions were manually defined with the help of the fibronectin staining in each individual plane along the Z axis. Areas negative for fibronectin signal were considered as tumor regions. Further data analysis was performed using Python and the scikit-image library. Images were first corrected for sample drift by detecting matching features in subsequent frames of the ECM channel and estimating transformation parameters based on their coordinates. Cells were segmented by intensity after background correction to reduce bleed-through of signal from the ECM channel and median filtering for smoothing. Detected features were filtered by size to remove noise and cell clumps. To facilitate tracking, cells that were apparent in more than one Z plane were only considered in the plane in which their size was maximal. Subsequent tracking of the detected cells was performed using Trackpy, and two tracks were merged if their respective initial or final point were less than 10 mm and less than two frames apart. Only tracks with data for at least six frames were considered for further analysis.
For localization analysis, cells were considered to localize to tumor regions when no pixel of the gd T-cell CMFDA signal overlapped with the fibronectin signal. A cell was defined to have a tumor dwell time >50% if it spent at least 50% of its observed frames localized to tumor regions. For speed analysis, the momentary speed of a cell was defined as the distance travelled between two successive observations of the same cell, divided by the time between these observations. Representative microscopy images and videos were generated using Imaris 9.3.1.

Preparation of tumor cell-derived conditioned medium
For transwell migration assays, conditioned medium (CM) was generated by harvesting the supernatant of BCSC cultures after 5 days. Culture supernatants were cleared from residual cells by centrifugation and stored at À80 C until use.
To generate CM from xenograft-derived cells, 5 Â 10 6 tumor cells were cultured in gd T-cell medium for 24 hours at 37 C in a humidified atmosphere of 5% CO 2 . Culture supernatants were cleared from residual cells by centrifugation and directly used for experiments.

Transwell and matrigel migration assays
For transwell migration assays toward CM, the CM was diluted 1:1.67 in gd T-cell medium/1% FBS. A total of 250 mL were transferred to the receiver wells of a 96-well transwell plate (Corning). Medium without cells, incubated in the same conditions as BCSCs, served as negative control. For migration toward the chemokine CXCL12, gd Tcell medium/1% FBS was supplemented with 50 ng/mL CXCL12 (PeproTech, 300-28A).
For transwell assays toward tumor cells, tumor cells were labeled with 20 mmol/L cell proliferation dye eFluor450 (Thermo Fisher Scientific). A total of 1.5 Â 10 5 tumor cells in 250 mL gd T-cell medium/1%FBS were seeded into the receiver wells of a 96-well transwell plate. gd T-cell medium/1% FBS served as negative control.
A total of 2 Â 10 5 gd T cells in 100 mL gd T-cell medium/1% FBS were seeded in the well of the permeable support with 5-mm pore size (Corning). After 3 hours incubation at 37 C, transmigrated cells were stained to distinguish T-cell subsets and counted via flow cytometry.
Matrigel migration assays were performed as described previously (25,26). Briefly, 3.5 Â 10 4 gd T cells were resuspended in 10 mL icecold Matrigel (Corning, 354234) and plated into the inner well of a precooled m-Slide Angiogenesis (ibidi). After cell settling at 4 C, samples were solidified at 37 C. A total of 15 mL Matrigel were added on top of the cell-containing gels. Following solidification at 37 C, wells were filled with gd T-cell medium supplemented with 10% FBS, 30 U/ml IL2, and 50 ng/mL CXCL12. After 48 hours, samples were fixed with 4% paraformaldehyde/0.25% glutaraldehyde for 20 minutes at room temperature. Gels were washed with PBS and cells were permeabilized with 0.5% Triton-X100 (Carl Roth) in PBS for 40 minutes. After washing, nuclei were stained with 5 mg/mL Hoechst 33342 (Sigma-Aldrich, 14533) for 3 hours in the dark and samples were imaged using confocal microscopy.
Images were acquired using a C2 confocal microscope (Nikon) with a 20Â objective (NA: 0.75, WD: 1 mm). Hoechst 33342 was excited with a 405 nm laser. Z stacks with a step size of 2 mm were imaged. Quantification of the migration distance was performed by using Python and the scikit-image library. Briefly, the voxels of the stacks were segmented into foreground (cells) and background by intensity thresholding. Overlapping cells were then separated in 3D with the Watershed algorithm. The position of a cell was defined to coincide with its centroid. Migration distances were normalized between images by baseline subtraction per image: the baseline migration distance for an image was defined as the 10th percentile of all cells' Z coordinates. This value was subtracted from all Z coordinates. Z coordinates were set to 0 if they became negative after baseline correction.

Sample preparation
BCSC5 culture cells and xenograft-derived tumor cells were washed with PBS, pelleted, frozen in liquid nitrogen, and stored at À80 C until further processing. Cell pellets were lysed in 400 mL lysis buffer [4% sodium dodecyl sulfate, 50 mmol/L tetraethylammonium bromide (pH 8.5), and 10 mmol/L tris(2-carboxyethyl)phosphine hydrochloride]. Lysates were boiled for 5 minutes and then sonicated for 15 minutes at high intensity (30 seconds on/30 seconds off). After sonication, DNA and RNA were degraded using Benzonase endonuclease (Sigma/Merck, E1014). The protein concentration was measured with EZQ Protein Quantitation Kit (Thermo Fisher Scientific, EZQ R33200). Lysates were alkylated in the dark with 20 mmol/L iodoacetamide for 1 hour at room temperature. For protein clean-up, 200 mg SP3 paramagnetic beads were added to the lysates, and proteins were bound to the beads by adding acetonitrile with 0.1% formic acid. Beads were washed in 70% ethanol and 100% acetonitrile before elution in digest buffer [0.1% sodium dodecyl sulfate, 50 mmol/L tetraethylammonium bromide (pH 8.5), and 1 mmol/L CaCl 2 ] and digested sequentially with LysC (Wako), then Trypsin (Promega, V5280), each at a 1:100 w/w (enzyme:protein) ratio. Peptide cleanup was performed according to the SP3 protocol.
Tandem mass tag labeling and basic C18 reverse phase chromatography fractionation Each sample (200 mg of peptides each) was resuspended in 100 mL of 100 mmol/L tetraethylammonium bromide buffer. TMT 10-plex (Thermo Fisher Scientific, 90110) labeling was performed according to manufacturer's protocol. To ensure complete labelling, 1 mg of labeled samples from each channel was analyzed by LC/MS-MS prior to pooling. The mixture of TMT 10-plex sample was desalted with Sep Pak C18 cartridge (Waters), and then fractionated by basic C18 reverse phase chromatography as described previously (27).

LC/MS-MS analysis
The LC separations were performed as described (27) with a Thermo Dionex Ultimate 3000 RSLC Nano liquid chromatography instrument. Approximately 1 mg of concentrated peptides (quantified by NanoDrop) from each fraction were separated over an EASY Spray column (C18, 2 mm, 75 mm Â 50 cm) with an integrated nanoelectrospray emitter at a flow rate of 300 nL/minute. Peptides were separated with a 180-minute segmented gradient. Eluted peptides were analyzed on an Orbitrap Fusion Lumos (Thermo Fisher Scientific) mass spectrometer.

Data analysis
All the acquired LC/MS-MS data were analyzed using Proteome Discoverer software v.2.2 (Thermo Fisher Scientific) with Mascot search engine. A maximum missed cleavages for trypsin digestion was set to 2. Precursor mass tolerance was set to 20 ppm. Fragment ion tolerance was set to 0.6 Da. Carbamidomethylation on cysteine and TMT 10-plex tags on N termini as well as lysine (þ229.163 Da) were set as static modifications. Variable modifications were set as oxidation on methionine (þ15.995 Da) and phosphorylation on serine, threonine, and tyrosine (þ79.966 Da). Data were searched against a complete UniProt Human (Reviewed 20,143 entries downloaded Nov 2018). Peptide spectral match error rates with a 1% FDR were determined using the target-decoy strategy coupled to Percolator modeling of true and false matches.
Both unique and razor peptides were used for quantitation. Signalto-noise (S/N) values were used to represent the reporter ion abundance with a coisolation threshold of 50% and an average reporter S/N threshold of 10 and above required for quantitation from each MS3 spectra to be used. The summed abundance of quantified peptides were used for protein quantitation. The total peptide amount was used for the normalisation. Protein ratios were calculated from medians of summed sample abundances of replicate groups. SD was calculated from all biological replicate values. The standard deviation of all biological replicates lower than 25% was used for further analyses.
Differentially regulated proteins were identified using a linear-based model (limma) on the normalized log 2 protein abundance. P value < 0.05 threshold was used as significance threshold. The Generally Applicable Gene-set Enrichment was used to retrieve the enriched processes. Several databases from MSigDB were used including Hallmark, Reactome, Gene Ontology, and immunologic signatures gene sets. P < 0.05 was used as significance threshold.

IP and Western blot analysis
Cells or xenografted tumors were lysed for 20 minutes on ice in lysis buffer [0.1% Nonidet P-40, 50 mmol/L HEPES (pH 7.0), 250 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.5 mmol/L dithiothreitol]. Lysis was followed by a 15-minute centrifugation to pellet the nuclei and insoluble materials. The supernatants were subsequently used as indicated. For HMG-CoA reductase (HMGCR) IP, 2 mg of anti-HMGCR antibody together with 10 mL of a mixture of 1:1 protein A and G coupled sepharose beads (GE Healthcare, 17513801 and 17061801) were added to lysates and incubated overnight at 4 C. Proteins from lysate or IP were subjected to SDS-PAGE followed by immunoblotting according to standard procedures (see Antibodies and chemicals). Protein bands were detected by chemiluminescence under a CCD camera (ImageQuant LAS 4000; GE Healthcare). Relative band intensity was quantified by ImageJ software and ImageQuantTL software (GE Healthcare).

HMGCR activity assay
The enzymatic activity of HMGCR was evaluated by quantitation of the NADPH extinction using the HMG-CoA reductase assay kit (Sigma-Aldrich, CS1090) with the samples generated for Western blot analysis using 50 mg total protein per reaction. A recombinant HMGCR provided by the kit was used as a positive control, the recombinant enzyme incubated with 5 mL of the Pravastatin (provided in the kit) was used as negative control, and only lysis buffer was used as blank. Samples, buffers, and substrates were added following the order in the manufacturer's protocol. Absorbance was measured at 340 nm using a microplate reader. The activity is expressed as AU/mg protein where 1 unit (AU) is the amount of HMGCR oxidating 1 mmol of NADPH in a minute at 37 C.

Statistical analysis
All data were tested for normality applying the D'Agostino and Pearson or Shapiro-Wilk test. For the comparison of two groups, an unpaired two-tailed Student t test was applied. For data not meeting the criteria for normality, the Mann-Whitney or Wilcoxon signed-rank test was applied. For the analysis of more than two groups, one-way ANOVA was applied in case of normally distributed data. Correction for multiple comparisons was performed by Dunnett test (comparing all groups with one control group). Nonparametric data were analyzed using the Kruskal-Wallis test followed by Dunn test (comparing all groups with one control group) to correct for multiple comparisons. Grouped analyses were tested using two-way ANOVA. To correct for multiple comparisons, Dunnett (comparing groups with respective control group inside of one row), Sidak (comparing groups with respective control group inside of one column or comparing two groups in one row), or Tukey (comparing all groups with each other) test was applied. If data were analyzed compared with a hypothetical value of 1 or 100, we used the one-sample t test for normally distributed data and the one-sample Wilcoxon test when nonparametric testing was suggested. log-rank test (Mantel-Cox) was used to calculate significance of differences between survival curves. Statistical analysis was performed using GraphPad Prism (v9, GraphPad Software). Applied analyses and statistical significances are indicated in the corresponding figures and figure legends. All data are represented as means AE SEM. Differences with P ≤ 0.05 were considered statistically significant. ns, nonsignificant; Ã , P ≤ 0.05; ÃÃ , P ≤ 0.01; ÃÃÃ , P ≤ 0.001; ÃÃÃÃ , P ≤ 0.0001. All data values and the corresponding statistical tests of each graph are available.

The Cancer Genome Atlas Analysis
The Cancer Genome Atlas (TCGA) RNA sequencing (RNA-seq) data were downloaded from TCGAbiolinks R/Bioconductor package. Only primary tumor samples from TNBC were retained in the analysis. In total, 180 RNA-seq samples with survival data available were analyzed. A list of TCGA barcodes is provided in Supplementary Table S1. Downstream analysis was performed with R (v4.2.2). Read counts were normalized by the library size (count per million). Genes quantified in less than 75% of the dataset were filtered out. Survival analysis, including Cox proportional hazard, was performed with "survival" and "survminer" R package.

Data availability
The proteomic data generated in this study are publicly available via ProteomeXchange with identifier PXD039463. Other data generated in this study are available within the article and its Supplementary Data or from the corresponding author upon reasonable request.

Expanded gd T cells efficiently kill patient-derived triplenegative BCSCs
To test the cytotoxic potential of human gd T cells toward patientderived triple-negative BCSCs, we expanded gd T cells from PBMCs of healthy donors using concanavalin A (ConA) stimulation. As described previously (23), expansion resulted in a specific enrichment of effector memory Vd1 þ and Vd2 þ T cells (Supplementary Fig. S1A). The expression of the activating natural killer-cell receptor NKG2D, which mediates the cytolytic activity of gd T cells (28), was upregulated during gd T-cell expansion (Supplementary Fig. S1B). We observed significantly higher NKG2D expression in Vd2 þ compared with Vd1 þ T cells during expansion; however, NKG2D expression was comparable at the end of the culture period (28 days). It has been reported that the overt expansion of ab T cells, their genetic modification or their stimulation after transfer to a patient can result in T-cell exhaustion and functional failure (29). Therefore, we followed the expression of the inhibitory receptors PD-1, TIM-3, and LAG-3 throughout the expansion (Supplementary Fig. S1B). PD-1 was upregulated within the first 10 days after ConA stimulation and then declined to basal levels. TIM-3, in contrast, exhibited a constant increase in expression as the expansion progressed. LAG-3 expression was drastically increased in the first 10 days after ConA stimulation, was then reduced but remained upregulated until the end of the expansion. The three inhibitory receptors were expressed at significantly higher levels in Vd2 þ compared with Vd1 þ T cells upon stimulation, and TIM-3 and LAG-3 remained highly expressed in Vd2 þ T cells at the end of the observed expansion period. Of note, the percentage of Vd1 þ and Vd2 þ T cells strongly varied between healthy donors. In the majority of donors, Vd2 þ T cells were highly enriched ( Supplementary Fig. S1C).
Expanded gd T cells were used for experiments between day 14 and day 35 after starting the expansion because the cells have reached a stable phenotype at this time window and cytotoxicity remained stable ( Supplementary Fig. S1D).
Next, we determined whether these gd T cells could kill patientderived triple-negative BCSCs in vitro. Expanded gd T cells from 3 healthy donors killed all tested patient-derived triple-negative BCSC lines (BCSC1, BCSC3, and BCSC5) in an effector to target ratiodependent manner (Fig. 1A). BCSC5 was most efficiently killed. To test whether the cytotoxicity was mediated by Vd1 þ or Vd2 þ T cells, we separated these two T-cell subsets after expansion ( Supplementary  Fig. S1D). Both Vd1 þ and Vd2 þ T cells displayed similar cytotoxicity in response to BCSC1, BCSC3, and BCSC5 (Fig. 1B). BCSCs only induced degranulation in Vd2 þ T cells, measured indirectly via CD107a (Fig. 1C). This was most prominent for BCSC5, which led to significantly higher degranulation in Vd2 þ T cells compared with BCSC1 and BCSC3. That Vd1 þ T cells did not degranulate after BCSC contact was not a consequence of a general incapability of Vd1 þ T cells to degranulate because stimulation with a-CD3 and a-CD28 antibodies led to CD107a accumulation in both T-cell subsets (Fig. 1C). These results suggest that Vd1 þ and Vd2 þ T cells kill patient-derived human BCSCs using different mechanisms.

Expanded gd T cells are attracted by BCSC CM
In vivo, in addition to recognizing and killing BCSCs, gd T cells need to be attracted and migrate toward the tumor sites. Therefore, we next assessed the migratory capacity of gd T cells toward BCSC CM in transwell experiments. CM from BCSC1, BCSC3, and BCSC5 efficiently attracted gd T cells (Fig. 2A). The migration of Vd1 þ T cells toward BCSC CM was increased compared with control medium, but strongly dependent on the T-cell donor. In contrast, Vd2 þ T cells from all tested healthy donors migrated robustly in response to BCSC CM (Fig. 2B). gd T cells outperformed ab T cells regarding their migration ability toward BCSC5 CM (Fig. 2C).
The different migratory responses exhibited by the specific T-cell subsets were more thoroughly investigated by analyzing the expression levels of 12 chemokine receptors (CXCR1, CXCR3, CXCR4, CXCR5, CXCR6, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR10). Among those, we found four of them, namely CXCR4, CXCR6, CCR4, and CCR7, to be differentially expressed in ab, gd, Vd1 þ , and Vd2 þ T cells (Fig. 2D). The expression of CXCR6 correlated well with the migratory responses observed. The only known ligand for CXCR6 is CXCL16, and CXCL16 was indeed detected in a proteomic approach using BCSC5 culture cells (Fig. 2E). Besides CXCL16, we identified the chemokines CXCL12, CXCL5, CXCL14, CCL20, and CCL28 to be expressed by BCSC5 (Fig. 2E). However, the chemokine expression pattern alone might be insufficient to identify the chemokine-chemokine receptor pairs involved in the attraction of T cells by BCSC CM due to the high degree of promiscuity defining the chemokine system. For example, even migration toward the well-known T-cell attractant CXCL12 only partially correlated with the expression levels of its best-characterized receptor CXCR4 ( Supplementary Fig. S2A and S2B).
Taken together, ConA-expanded gd T cells most efficiently recognized and killed BCSC5 among the tested BCSCs, and exhibited Expanded gd T cells migrate toward BCSC CM. A, Schematic transwell migration assay (left). Primary T cells were seeded in the wells of a permeable support with 5-mm pore size. The lower compartment was filled with BCSC CM and cells that transmigrated into the lower compartment were analyzed by flow cytometry. Statistical analysis of transmigrated gd T cells (CD3 þ gdTCR þ ) is shown (right). Basal migration toward medium was set to 1.0 and fold changes from two independent experiments using 7 healthy donors in total were pooled (median, minimum to maximum). One-sample t test against hypothetical value of 1.0. B, Transwell assays were performed and analyzed as in A. Transmigrated Vd1 þ (CD3 þ gdTCR þ Vd1 þ ) and Vd2 þ (CD3 þ gdTCR þ Vd2 þ ) T cells were distinguished by flow cytometry. Onesample t test (for Vd1 þ ) or one-sample Wilcoxon test (for Vd2 þ ). C, Transwell assays were performed and analyzed as in A. Comparison of transmigrated ab T cells (CD3 þ gdTCR À ) and gd T cells (CD3 þ gdTCR þ ; data from A) in response to BCSC5 CM. One sample t test against hypothetical value of 1.0. D, Representative chemokine receptor expression levels in ab (CD3 þ gdTCR À ), gd (CD3 þ gdTCR þ ), Vd1 þ (CD3 þ gdTCR þ Vd1 þ ), and Vd2 þ (CD3 þ gdTCR þ Vd2 þ ) T cells. Shown are representative flow cytometry histograms from one healthy donor out of 3 healthy donors analyzed. E, Chemokine expression levels determined by quantitative mass spectrometry of BCSC5. Protein intensities (log 2 ) of three replicates are shown (means AE SEM). Ã , P ≤ 0.05; ÃÃ , P ≤ 0.01; ÃÃÃ , P ≤ 0.001.

Breast Cancer Stem Cells Escape gd T-cell Immunosurveillance
AACRJournals.org Cancer Immunol Res; 11(6) June 2023 a robust migration toward BCSC5 CM. Therefore, we focused our studies on BCSC5 and conducted further experiments with gd T-cell cultures containing mainly Vd2 þ T cells and less than 10% of Vd1 þ T cells to minimize heterogeneity.

MMP14 expression in gd T cells increases their migration capacity in ECM-rich environments
Our transwell assays demonstrated that gd T cells were efficiently attracted toward BCSC5 CM. However, BCSC-derived xenotransplanted tumors are surrounded by a dense ECM, similar to the original TNBC tumors (22). This ECM-rich stromal compartment might hamper gd T-cell migration and infiltration. Therefore, we aimed to boost gd T-cell migration by expressing the membrane-anchored MMP14 in gd T cells. MMP14 is one of 26 known endopeptidases of the human MMP protein family (30). It can cleave a plethora of ECM proteins like fibronectin, collagen (type I, II, and III), and laminin (31,32). Furthermore, MMP14 has been intensively studied in the process of tumor-cell migration where it was shown to possess promigratory functions (33). We found that MMP14 was endogenously expressed in gd T cells directly after ConA stimulation but was downregulated over time. In contrast, ab T cells did not upregulate MMP14 upon activation ( Supplementary Fig. S3A). To maintain MMP14 expression, expanded gd T cells were lentivirally transduced with a mock vector, MMP14, or the catalytically inactive mutant MMP14 E240A , and expression was verified by flow cytometry (Fig. 3A; Supplementary Fig. S3B). We then assessed the effect of MMP14 on gd T-cell migration towards CXCL12 and FBS in Matrigel, a model matrix for basement membranes (Fig. 3B). MMP14 expression increased the percentage of gd T cells migrating further than 10 or 100 mm, while the catalytically inactive mutant failed to promote migration (Fig. 3B). These findings demonstrate that MMP14 has the potential to promote the migration of gd T cells in basement membrane-like ECM.
To test whether the promigratory function of MMP14 observed in Matrigel also supports interstitial migration in tumors, we analyzed gd T-cell migration in viable slices of BCSC5 xenograft tumors via confocal microscopy following published protocols (Supplementary Fig. S3C; ref. 18). For this purpose, BCSC5 cells were orthotopically transplanted into the mammary fat pad of NOD SCID mice as described previously (22,34,35). CMFDA-labeled nontransduced gd T cells or gd T cells expressing MMP14 were plated on top of unfixed xenograft-derived tumor slices and were microscopically monitored. EpCAM and fibronectin staining served to distinguish tumor islets and ECM-rich stroma, respectively [ Fig. 3C; Supplementary Videos S1 and S2, for untransduced (UT) and MMP14, respectively]. MMP14 expres-sion increased the interstitial migration speed of gd T cells when compared with nontransduced cells (Fig. 3D, left). When differentially analyzing the migration speed of gd T cells within the tumor tissue or the stromal ECM, we observed that nontransduced gd T cells migrated faster in the tumor tissue than in the stromal ECM (Fig. 3D). This is in line with observations made for ab T cells in ovarian and lung cancer (19). MMP14 expression increased the average speed of gd T cells in the ECM to the speed exhibited in the tumor tissue, supporting the functional role of MMP14 in cleaving the tumorassociated ECM (Fig. 3D, right). In addition, a higher percentage of cells per time-lapse experiment was in direct tumor contact when the cells expressed MMP14 (Fig. 3E) and a bigger fraction of gd T cells resided in the tumor for more than 50% of their monitored time when expressing MMP14 (Fig. 3E, right). Altogether, these results suggest that the protease MMP14 can boost the migration of gd T cells in the ECM-enriched tumor environment, and might thereby help to overcome limitations that hamper gd T-cell migration in vivo and, ultimately, therapy outcome.

gd T cells fail to control BCSC5 orthotopic xenografts
After establishing a system to maximize gd T-cell targeting of BCSC ex vivo, we assessed whether gd T cells could control BCSC5 tumor growth in vivo. To this end, we generated BCSC5 orthotopic mammary gland xenografts in NOD SCID mice as described previously (22,34,35). Mice were treated with a vehicle control, gd T cells, or gd T cells expressing MMP14 once the tumors had reached a volume of at least 4 mm 3 (Supplementary Fig. S4A). The median survival of the mice after treatment started increased from 28 days for vehicle-treated animals to 33 and 38 days for gd T cell-treated or gd T cell MMP14treated mice, respectively (Fig. 4A). Although a clear tendency toward increased survival times of the mice treated with gd T cells or gd T cells expressing MMP14 was observed, there was no significant increase in the overall survival between the different treatment groups. Furthermore, the treatment did not significantly affect the growth kinetics of the individual tumors, although a higher proportion of tumors slowed down their growth when MMP14 was expressed in gd T cells (Fig. 4A,  right). In addition, neither treatment with gd T cells nor with gd T cells expressing MMP14 increased tumor cell invasion or metastasis when compared with vehicle-treated animals. Similar results were obtained using alternative immunocompromised mice, namely Rag2 À/À gc À/À (Supplementary Fig. S4B).
These results raised the question as to whether BCSC5 xenograftderived tumor cells could still be recognized and killed by gd T cells. Thus, we isolated tumor cells from vehicle-treated, gd T cell-treated or gd T cell MMP14-treated mice after tumor resection and analyzed the degranulation capacity of gd T cells in response to these xenograftderived tumor cells (indicated as "xeno" in the figures). We observed that the proportion of degranulating gd T cells was significantly reduced from 75% to approximately 30% when compared with the response toward in vitro cultured BCSC5 (Fig. 4B, left). Similarly, we observed reduced gd T cell-mediated killing of xenograft-derived tumor cells compared with BCSCs from the culture (Fig. 4B, right). These effects were inherent to the tumor cells and not influenced by the gd T-cell treatment of the tumor-bearing mice (Fig. 4B). We also determined whether secreted soluble molecules decreased the response of gd T cells to xenograft-derived tumor cells. We analyzed gd T-cell viability and gd T-cell functionality after culturing the cells in CM from BCSC5 culture cells or xenograft-derived tumor cells for 24 hours as described previously (17). gd T-cell viability was not influenced by CM from either cell type (Supplementary Fig. S4C). Likewise, neither degranulation nor the cytotoxic response of gd T cells to BCSC5 was affected by the CM (Supplementary Fig. S4D and S4E). These findings indicate that the reduced gd T-cell function in response to xenograftderived tumor cells was not mediated by the secretion of soluble immunosuppressive molecules. As mentioned before, triggering of inhibitory receptors such as PD-1 by their ligands can result in the functional inhibition of T cells. We observed that the ligands for the inhibitory receptor PD-1, PD-L1, and PD-L2, were upregulated on xenograft-derived tumor cells (Fig. 4C). To assess whether PD-1 blockade improved gd T-cell treatment in vivo, we combined the adoptive transfer of gd T cells with biweekly applications of a clinically relevant anti-PD-1 antibody (nivolumab). However, the combination treatment did not improve the overall survival of the xenograft-bearing mice but showed a slight reduction in the tumor growth kinetics in vivo (Fig. 4D  and E). These results suggest that blocking PD-1 was not sufficient to significantly induce gd T cell-mediated tumor rejection and that other mechanisms might be involved in the immune escape of BCSC5-derived xenografts.

gd T cells are efficiently attracted by xenograft-derived tumor cells
To discover proteins and mechanisms involved in the xenograft immune escape in vivo, we utilized LC/MS-MS to compare the xenograft-derived tumor cell proteomes of all treatment groups with the proteome of BCSC5 culture cells. These analyses revealed that tumor growth in NOD SCID mice drastically changed the proteome of the cells. Around 5,600 proteins were differentially expressed by vehicle-treated xenograft cells compared with BCSC5 culture cells (Fig. 5A). Next, we more closely examined the chemokine secretion profiles of xenografted BCSC5 compared with culture cells to exclude the possibility that xenograft-derived cells lost their ability to attract gd T cells to the tumor site in vivo. Of the detected chemokines, CXCL5 expression was reduced in the xenograft-derived tumor cells compared with BCSC5 culture cells, while CCL20 and CCL28 expression was augmented (Fig. 5B). The rest of the detected chemokines remained unchanged (CXCL12, CXCL14, and CXCL16). None of the observed changes could be associated with a specific treatment. Next, we analyzed gd T-cell migration toward BCSC5 culture cells and freshly isolated xenograft-derived tumor cells. Indeed, cells derived from the xenograft attracted gd T cells more efficiently than BCSC5 culture cells (Fig. 5C). We assayed Vd1 þ and Vd2 þ T cells separately and observed that Vd2 þ T cells migrated more efficiently toward cells derived from the xenograft compared with Vd1 þ T cells (Fig. 5D). These results highlight that xenografts escaped gd T-cell immunotherapy by other means than reducing gd T-cell attraction.

BCSC5 cells differentiate in vivo and downregulate the expression of gd T-cell ligands
Next, we performed pathway analyses on our proteomic data and verified that BCSC5 culture cells exhibited a mammary stem cell (MSC) signature. However, proteins usually expressed by MSCs were significantly downregulated in xenograft-derived tumor cells and, vice versa, proteins usually lowly expressed in MSCs were upregulated (Fig. 6A). These findings indicated that BCSC5 cells differentiated in vivo losing their breast stem cell signature, despite BCSC-derived cells preserving the patient's original molecular tumor subtype (22,34).
In addition to changes affecting stemness, the transfer and growth of BCSC5 in vivo drastically changed the expression levels of well-known gd T-cell ligands and other molecules involved in cancer cell recognition by gd T cells (Fig. 6B). We found that a variety of these proteins were specifically downregulated in xenograft-derived tumor cells and that these changes were independent of the gd T-cell treatment (Fig. 6B). We validated the reduced expression of ULBP-2/5/6, Fas, and ICAM-1 via flow cytometry (Fig. 6C). All these proteins have been previously described to be involved in tumor cell killing by gd T cells (13,(36)(37)(38). Although TRAILR1 and TRAILR2 were not identified in our proteomic study, TRAIL-TRAILR interactions induce gd T cell-mediated killing (39). While TRAILR1 expression was not significantly changed on xenograft-derived tumor cells compared with BCSC5 culture cells, TRAILR2 levels were drastically reduced . gd T cells fail to control BCSC5 xenografts in NOD SCID mice. A, Kaplan-Meier plot (left) of BCSC5 xenograft-bearing mice upon treatment with gd T cells (blue), gd T cells expressing MMP14 (red) or vehicle control (black; n ¼ 6-7 mice per group). Differences were not statistically significant, log-rank test (Mantel-Cox). BCSC5 tumor growth curves (right) for individual mice after treatment start. B, gd T cell-mediated degranulation (left) and cytotoxicity (right) of BCSC5 culture cells or xenograft-derived tumor cells (xeno). Xenograft-bearing mice were treated with gd T cells, gd T cells expressing MMP14 or vehicle. For the degranulation assay, gd T cells were cocultured with the respective tumor cells for 3 hours. The percentage of CD107a þ cells of Vd2 þ -gated cells is shown (means AE SEM). Four to six tumors were analyzed per group and each tumor was tested with 2 healthy donors of gd T cells. For in vitro killing of 51 Cr-labeled tumor cells, gd T cells were cocultured with the target cells for 5 hours at an E:T ratio of 30:1. Results from three independent experiments with a total of 3 healthy donors of gd T cells and three tumors per group were pooled (means AE SEM). One-way ANOVA followed by Dunnett post hoc test comparing xenograft with culture cells. C, Flow cytometry-based analysis of PD-L1 and PD-L2 expression levels in xenograft-derived EpCAM þ tumor cells (median, minimum to maximum). Mean fluorescence intensity (MFI) of 9-14 tumors per group is shown. Kruskal-Wallis test followed by Dunn post hoc test comparing xenograft with culture cells. D, Kaplan-Meier plots of NOD SCID mice upon treatment with vehicle control (black), gd T cells (blue) or gd T cells expressing MMP14 (red) in combination with an anti-PD-1 (nivolumab) or isotype control antibody (n ¼ 5-6 per group). Treatment start was defined for each mouse individually when the first tumor reached a volume of at least 4 mm 3 . A total of 5 Â 10 6 gd T cells were injected intravenously three times per week. In addition, mice received 0.6 Â 10 6 IU IL2 (Proleukin S) on the day of treatment start and every 21 days until the end of the experiment. The end of the experiment was defined by a tumor volume of 800 mm 3 . No significant differences were obtained, log-rank (Mantel-Cox) test. E, BCSC5 tumor growth curves for individual mice upon treatment with gd T cells (blue), gd T cells expressing MMP14 (red) or vehicle control (black) in combination with anti-PD-1 or isotype control antibody (n ¼ 5-6 per group). Ã , P ≤ 0.05; ÃÃ , P ≤ 0.01; ÃÃÃ , P ≤ 0.001.

Breast Cancer Stem Cells Escape gd T-cell Immunosurveillance
AACRJournals.org Cancer Immunol Res; 11(6) June 2023 (Fig. 6C). Taken together, the loss of the above-mentioned proteins might explain why gd T cells cannot efficiently recognize and kill xenograft-derived tumor cells. We next analyzed TCGA (https://www. cancer.gov/tcga) datasets for patients with TNBC scoring for the expression level of Fas, MICA/B, TRAILR1/2, and ICAM-1 individually. Cox proportional hazards analysis failed to reveal significant prolonged survival for patients with high expression of each of the analyzed proteins ( Supplementary Fig. S5). This analysis suggests that each of these proteins individually cannot predict suitability for eventual immunotherapies using gd T cells. Next, we aimed to elucidate the mechanisms involved in the killing of BCSC5 by gd T cells to narrow down which phenotypic changes might be mainly responsible for the immune escape of xenografted BCSC5. We investigated the role of Fas, MICB, TRAILR1/2, and ICAM-1 in the recognition and subsequent killing of BCSC5 by performing cytotoxicity experiments using blocking antibodies. We found that gd T cell-mediated killing of BCSC5 involves Fas-FasL interactions, MICA/B engagement and, most drastically, ICAM-1 binding (Fig. 6D). In contrast, TRAIL-TRAILR interactions, ULBP2/5/6 engagement, and the binding of CD103 (integrin a E) did not play a major role in BCSC5 killing (Fig. 6D). These results indicate that among the phenotypic changes observed upon xenotransplantation, the loss of Fas, MICA/B and ICAM-1 expression might play a crucial role in protecting these cells from gd BCSCs phenotypically change in vivo, but still induce gd T-cell migration. A, The proteomes of BCSC5 culture cells or freshly isolated xenograft-derived tumor cells were analyzed by mass spectrometry. Volcano plot showing differentially expressed proteins. Proteins regulated with P < 0.05 are depicted in red. B, Rowwise Zscore heat maps showing upregulated and downregulated proteins in BCSC5 culture cells or xenograft-derived tumors from two or three biological replicates (BR) as indicated. C, Migration of gd T cells (CD3 þ gdTCR þ ) in response to BCSC5 culture cells and xenograft-derived tumor cells was determined in a transwell assay. Basal migration towards medium was set to 1.0 and fold changes from three independent experiments using the same 3 healthy donors in each experiment were pooled and analyzed using one-sample Wilcoxon test. D, Migration of Vd1 þ (CD3 þ gdTCR þ Vd1 þ ) and Vd2 þ (CD3 þ gdTCR þ Vd2 þ ) T cells in response to BCSC5 culture cells and xenograft-derived tumor cells was determined in a transwell assay. Basal migration toward medium was set to 1.0 and fold changes from three independent experiments using the same 3 healthy donors in each experiment were pooled. One-sample t test (for Vd1 þ ) or one-sample Wilcoxon test (for Vd2 þ ). Ã , P ≤ 0.05; ÃÃ , P ≤ 0.01; ÃÃÃÃ , P ≤ 0.0001. Non-adj., non-adjusted.
T cell-mediated cytotoxicity. Our data also suggest that gd T cells might require multiple receptor-ligand interactions to efficiently kill BCSCs. We hence clustered TCGA data from patients with TNBC based on the level of expression of the three key proteins Fas, MICB and ICAM-1. Cox proportional hazards analysis showed that patients expressing high levels of these proteins have a 1.4 times lower 5-year mortality risk (Fig. 6E).

Zoledronate sensitizes xenograft-derived tumor cells to gd T cell-mediated killing
In addition to proteins directly involved in killing, we identified HMGCR to be significantly downregulated in the xenograft-derived tumor cells (Fig. 6B; Fig. 7A and B). The SREBP-SCAP complex regulates the transcription of HMGCR. Indeed, SREBP-SCAP protein levels were significantly reduced in xenograft-derived tumor cells ( Supplementary Fig. S6). Consequently, HMGCR transcripts were reduced both in the xenograft-derived tumor cells as well as in the original patient tumors indicating that in vivo differentiation of BCSCs reduces HMGCR both in mouse models and in patients (Supplementary Fig. S6). Furthermore, HMGCR activity was significantly reduced in lysates obtained from xenograft-derived tumor cells (Fig. 7C). HMGCR is the rate-limiting enzyme of the mevalonate pathway, which regulates the levels of pAgs in tumor cells (40). These pAgs can be recognized by the Vg9Vd2 TCR expressed by Vd2 þ T cells in the context of butyrophilin 3A1 (BTN3A1) and 2A1 (BTN2A1; refs. 41,42). Expression of BTN3A1, however, was not significantly altered in xenograft-derived tumor cells, and BTN2A1 was not detected in the proteomic study (Fig. 6B). Likewise, no changes were observed using an antibody recognizing all BTN3A isoforms (Fig. 6C).
Considering that the BTN3A1 levels were comparable between BCSC5 culture cells and xenograft-derived tumor cells, we hypothesized that the loss of HMGCR expression was responsible for unresponsiveness of gd T cells due to low pAg levels in the tumor cells. To further explore this, we performed cytotoxicity assays in the presence of zoledronate or mevastatin, two well-studied drugs interfering with the mevalonate pathway and modulating pAg levels (43,44). Zoledronate, most probably via the accumulation of pAgs, increased gd T-cell cytotoxicity toward xenograft-derived tumor cells (Fig. 7D). The cytotoxicity in the presence of zoledronate was similar to the killing of untreated BCSC5 culture cells (Fig. 7D). In contrast, the accumulation of pAgs by zoledronate in BCSC5 culture cells failed to increase gd T-cell cytotoxicity toward these cells, suggesting that this recognition axis was already saturated (Fig. 7D). Reducing pAg levels by mevastatin led to reduced killing of BCSC5 culture cells and almost completely abolished the killing of xenograft-derived cells (Fig. 7D). These findings show that the killing of xenograft-derived tumor cells by gd T cells almost exclusively relied on the recognition of pAgs and that these cells can be sensitized to gd T cell-mediated killing by zoledronate treatment. Both BTN2A1 and BTN3A1 are necessary for the recognition of pAgs by gd T cells (41,42). The correlation between the mRNA levels of BTN2A1 and BTN3A1 does not change between mammary healthy tissue (0.5988) and TNBC patient-derived samples (0.5924). We clustered patients with TCGA TNBC by the expression levels of both BTN2A1 and BTN3A1 and found that high expression of both proteins significantly correlated with increased survival. Patients with high levels of BTN2A1 and BTN3A1 mRNA had a two times lower 10-year mortality risk (Fig. 7E). BCSC5 culture cells, in contrast, can still be partially recognized in the presence of mevastatin, in line with the finding that BCSC5 can be killed by several mechanisms, namely those involving Fas, MICB, and ICAM-1 as detailed above (Figs. 7D and 6D). Thus, we clustered patients with TCGA TNBC by the mRNA levels of BTN2A1 and BTN3A1, which are key for the recognition of differentiated cells, and Fas, MICB, and ICAM-1, which seem to be central for the recognition and killing of BCSCs. Patients with high levels of these proteins showed significantly increased survival having a 1.4 times lower 5-year mortality risk (Fig. 7F). Gene set enrichment analysis of TCGA data revealed that patients with TNBC clustered by high (top quartile) average expression of BTN2A1, BTN3A1, Fas, MICB, and ICAM-1 exhibited higher expression of BCSC genes and gene signatures associated to immune response, inflammation, IFNg and IFNa responses, cytokine and chemokine signaling and immune-mediated cytotoxicity ( Supplementary Fig. S7) suggesting a favorable tumor immune microenvironment. To deepen into this observation, we applied the "deep deconvolution" CIBERSORT algorithm to TCGA data to deduce the immune cell composition (45) and compared the patients with TNBC with high versus low average expression of BTN2A1, BTN3A1, Fas, MICB, and ICAM-1 ( Supplementary  Fig. S8). CD4 þ memory T cells, resting and activated, CD8 þ T cells, and antitumor M1 macrophages were enriched in patients with TNBC with high average expression. In contrast, M2 tumor-promoting macrophages were significantly reduced. The assessment of tumorinfiltrating human gd T cells by deconvolution has proven to be challenging, therefore we analyzed genes specifically upregulated in human gd T cells that have been identified by machine learning approaches (46). Indeed, these human gd T cell-specific genes were clearly upregulated in those patients with TNBC clustered by high (top quartile) average expression of BTN2A1, BTN3A1, Fas, MICB, and ICAM-1 ( Supplementary Fig. S8). Thus, expression of these proteins might be an instrumental tool to identify patients with TNBC suitable for gd T cell-based immunotherapy approaches.
In addition to cytotoxicity, gd T cells play a critical role in protective immune responses against tumor development by providing an early source of the proinflammatory cytokine IFNg (16,47). IFNg plays a manifold role in activating anticancer immunity. For instance, IFNg promotes the activity of tumor-triggered ab T cells and inhibits the differentiation and activation of regulatory ab T cells (48). The current view is that IFNg-producing cells are endowed with potent cytotoxic functions during antitumor responses (49). In our settings, gd T cells, with or without zoledronate treatment, did not respond with IFNg secretion to xenograft-derived tumor cells (Fig. 7G), which might limit the protective immune response against tumor development. It has been previously shown that IFNa can mediate an increase in IFNg secretion by pAg-activated Vd2 þ T cells (50). In line with this report, the pretreatment of tumor cells with IFNa induced gd T cell-mediated IFNg secretion in response to xenograft-derived tumor cells (Fig. 7H) while it failed to rescue gd T cell-mediated cytotoxicity toward xenograft-derived tumor cells (Fig. 7I). Taken together, the treatment with IFNa facilitated the activation of gd T cells to produce IFNg, which might be therapeutically interesting in clinical settings in which tumor-triggered ab T cells play an important role. Yet, this boost of gd T cells to produce IFNg did not translate into increased cytotoxicity toward BCSCs highlighting that a distinct signals and/or a different threshold of activation are needed to produce IFNg or to induce killing by gd T cells. The molecular identification and targeting of the pathways regulating these two effector functions are beyond the scope of the current study.
In summary, our proteomic studies and biological validations strongly support the hypothesis that BCSC5 culture cells differentiated in vivo. This differentiation involved the upregulation of inhibitory Tcell receptors, the loss of stem cell characteristics, the reduction of numerous gd T-cell ligands and a decrease in pAg levels, in sum preventing efficient recognition and killing by gd T cells.

Discussion
It has been suggested that BCSCs are responsible for therapy resistance and metastatic dissemination in breast cancer, which is the leading cause of cancer deaths among women worldwide. Until now, treatment-resistant BCSCs have only been poorly characterized, and targeted therapeutics have yet to be identified. We have recently established an optimized culture system to expand human BCSCs that faithfully reproduce the original patient's tumor characteristics and are therefore an ideal cellular platform to test novel therapeutics (22,34). Here, we aimed to investigate the susceptibility of these BCSCs to immunotherapy using primary human gd T cells. We showed that patient-derived triple-negative BCSCs are targeted by both Vd1 þ and Vd2 þ primary expanded gd T cells. However, orthotopically xenografted BCSC5, the BCSC line best recognized by gd T cells in our study, was refractory to gd T-cell immunotherapy. We demonstrated that attraction and/or migration toward xenografted cells was not the reason for this unexpected in vivo outcome. Both Vd1 þ and Vd2 þ T cells migrated efficiently toward BCSC CM and this migration was even increased toward xenografted cells. The chemokine receptors expressed on the expanded gd T cells were compatible with the secretion profile of xenografted cells.
Solid tumors, including breast cancer, are often surrounded by a dense ECM preventing the efficient infiltration by immune cells (25). A high stromal content has been associated with poor prognosis in TNBC, and an accumulation of gd T cells in the tumor stroma contributes to this observation (51). Detailed analyses of gd T-cell migration with respect to the tumor stroma are not yet available. Here, we found that exogenously expressing MMP14 conferred promigratory functions to primary gd T cells in a 3D model for basement membranes. Crossing of the basement membrane is of critical relevance for gd T cells to extravasate from the blood stream at the tumor site (52). Using viable slices of BCSC5-derived xenograft tumors, we showed that gd T-cell migration was accelerated inside of tumor islets compared with the ECM-rich tumor stroma similar to previous observations made for ab T cells (19). MMP14 expression increased gd T-cell migration speed exclusively in the tumor stroma and not inside the tumor tissue, supporting the functional role of MMP14 in cleaving the peritumoral ECM (31). These findings are in line with a previous report showing that expressing the secreted ECM-modifying enzyme heparanase in human CAR ab T cells promoted tumor rejection in melanoma and neuroblastoma xenograft models (21). In another study, inhibition of the ECM cross-linking enzyme lysyl oxidase improved antitumor responses in combination with checkpoint inhibition due to increased ab T-cell migration and infiltration (53). We hypothesize that the overexpression of MMP14 might have certain advantages over the expression of secreted heparanase or the systemic application of lysyl oxidase inhibitors, as MMP14 is a membrane-anchored protein lowering the risk of structural changes outside of the tumor tissue. Although we have not observed any side effects related to MMP14 expression in mice receiving gd T-cell treatment, the application of MMP14 will have to be strictly monitored with respect to biodistribution and its impact on healthy tissues in future studies.
Despite increased chemoattraction and MMP14-mediated peritumoral migration, gd T cells failed to control BCSC5-derived tumors in vivo. Remarkably, xenografted cells showed reduced capacity to activate gd T cells and thereby reduced susceptibility to gd T cellmediated cytotoxicity. Despite our BCSCs reliably reproducing many original patient's tumor characteristics (22,34), they underwent major changes in their proteomic signature after xenograft and growth in vivo. BCSC5 cells lost their stem cell characteristics and downregulated a plethora of surface proteins key for immunosurveillance by gd T cells after transplantation and growth in vivo. Intriguingly, this in vivo differentiation was not a consequence of mechanisms induced by the immune system or the immunotherapy, because it was equally observed in immunodeficient mice with or without the presence of primary human gd T cells. Xenografted cells exhibited increased surface expression of the inhibitory T-cell ligands PD-L1 and PD-L2 compared with parental BCSC5 cells, in line with studies associating TNBC with high expression levels of PD-L1 (54). This increased expression of inhibitory T-cell ligands might explain the reduced susceptibility to be killed by gd T cells. However, a combinatorial treatment of xenografts with gd T cells and anti-PD-1 did not result in reduced xenograft growth. Similarly, Li and colleagues have described the inefficacy of this treatment regimen against TNBC MDA-MB-231derived xenografts (54).
Because increased PD-L1 levels were not responsible for the immune evasion of xenografted cells, we searched for additional molecules involved in BCSC recognition and compared their expression levels before and after in vivo growth. Our results revealed that recognition and killing of BCSC5 by gd T cells required the engagement of multiple receptor-ligand pairs. Killing was thus dependent on ICAM-1 binding, MICB, pAgs/BTN2A1/BTN3A1 and on Fas-FasL interactions. Indeed, clustering patients with TNBC for high expression of these proteins significantly improved survival prognosis for this group. In ab T cells, ICAM-1 is key for the formation of a functional immune synapse and to enable CAR ab T-cell entry into solid tumors (55). Low ICAM-1 expression levels on breast cancer cells made them resistant to ab T-cell killing (16). Likewise, human pancreatic cancer cell lines lacking ICAM-1 were poorly bound and killed by gd T cells in vitro (36). Thus, our results support that ICAM-1-mediated intercellular interactions facilitate gd T cellmediated recognition and killing of BCSCs by Fas-FasL interactions and pAgs. However, xenograft-derived tumor cells lost Fas, MICB, Figure 6. BCSCs differentiate in vivo and downregulate the expression of proteins recognized by gd T cells. A, Rowwise Z-score heat maps showing upregulated and downregulated proteins in BCSC5 culture cells or xenograft-derived tumors from two or three biological replicates as indicated (BR). B, Rowwise Z-score heat map for proteins involved in target cell recognition by gd T cells or in immune response modulation. C, Flow cytometry-based analysis of BCSC5 culture cells and xenograft-derived tumor cells. MFI of at least five tumors per group is shown (median, minimum to maximum). One-way ANOVA followed by Dunnett post hoc test comparing xenograft-derived cells with culture cells (for ULBP2/5/6, ICAM-1, and BTN3A). Kruskal-Wallis test followed by Dunn post hoc test comparing xenograftderived cells with culture cells (for Fas, TRAILR1, and TRAILR2). D, Killing of BCSC5 by gd T cells requires multiple ligand-receptors interactions. In vitro killing of luciferase-expressing BCSC5 cells by gd T cells. gd T cells or BCSC5 culture cells were pretreated with the indicated blocking antibodies for 1 hour. Cells were cocultured at an E:T ratio of 10:1 for 8 hours in the presence of the blocking antibodies or the corresponding isotype controls. Corresponding receptor-ligand pairs are represented in the same color. Isotype antibody control was set to 100% for normalization in each experiment. Results for 3 to 4 healthy donors of gd T cells from three independent experiments were pooled (means AE SEM). One-sample t test against hypothetical value of 100. E, Cox regression of progression-free survival for patients with TNBC sorted by high (top quartile) and low (bottom quartile) average clustered expression of the Fas, MICB, and ICAM-1. b Cox coefficient was À0.33 and the Cox P value was 0.07 suggesting that high expression of the three proteins correlates with a better survival prognosis. Ã , P ≤ 0.05; ÃÃ , P ≤ 0.01; ÃÃÃ , P ≤ 0.001; ÃÃÃÃ , P ≤ 0.0001. and ICAM-1 expression, and downmodulated HMGCR, the ratelimiting enzyme of the mevalonate pathway producing pAgs. These changes are most probably responsible for the escape of xenograftderived tumor cells from gd T-cell recognition. Yet we observed some residual cytotoxicity toward xenograft-derived tumor cells that was exclusively dependent on pAg recognition as pharmacologic inhibition of HMGCR by mevastatin abolished cytotoxicity. Accumulation of pAgs by zoledronate pretreatment overcame the resistance of xenograft-derived tumor cells to gd T cell-mediated killing. The clinical application of zoledronate is FDA approved for the treatment of osteoporosis and bone metastasis. Therefore, combinatorial therapy approaches using zoledronate and the adoptive transfer of gd T cells represent a promising option to simultaneously tackle BCSCs and their differentiated progeny. Our observation that BCSCs can be better recognized by gd T cells than their differentiated progeny apparently opposes previous reports using the expression of CD44 and CD24 to define stemlike cells (CD44 hi CD24 lo ) and their non-stem cell counterparts (CD44 hi CD24 hi ). Sorted stemlike cells from the triple-negative SUM149 cell line and from PDX401 cells were less efficiently killed by gd T cells compared with nonstem cell counterparts (17). This study identified MICA shedding from the tumor cell surface as a mechanism to escape gd T-cell recognition. MICA surface levels were unchanged between the differentiated xenograft cells and the BCSCs suggesting that MICA shedding does not play a major role in the escape of BCSC5-derived xenografts in vivo. To the best of our knowledge, only one other study investigated the response of gd T cells against BCSCs and nonstem cells derived from ras-transformed human mammary epithelial cells, and found that both cell populations were equally resistant to gd T cell-mediated killing (16). Yet, both populations could be sensitized by zoledronate treatment in line with our results. Taken together, these discrepancies among studies underline the importance of perceiving immunotherapeutic approaches as individualized medicine, which might have to be tailored for each patient.
In addition, our results highlight a previously unnoticed dichotomy, namely that IFNg responses and cytotoxicity by gd T cells do not necessarily correlate. Zoledronate enhanced cytotoxicity by gd T cells but failed to promote IFNg secretion. In contrast, IFNa treatment increased gd T cell-mediated IFNg secretion but failed to enhance gd T cell-mediated cytotoxicity. This result suggests that IFNa might represent an attractive tool to be used in combinatorial therapies to induce synergistic effects of gd and ab T-cell responses.
In summary, our data show that patient-derived triple negative BCSCs are targetable by expanded gd T cells. However, in vivo growth of these BCSCs leads to their differentiation into cells that lost stemness and ligands to activate gd T-cell responses and thereby, escaped from efficient killing by gd T cells. Still, gd T cells residually killed in vivo differentiated cells by recognizing pAgs. This killing could be increased to the level of BCSC5 killing by zoledronate. In all, we propose that a combinatorial therapy using gd T cells and zoledronate represents a valuable approach to target triple-negative BCSCs and non-stem cells alike. Furthermore, IFNa treatment could induce IFNg production by gd T cells, and thereby induce a first source of IFNg promoting ICAM-1 expression, T-cell entry into solid tumors (55) and further endogenous ab T-cell responses in immunocompetent settings.