Cyclophosphamide and Vinorelbine Activate Stem-Like CD8 T Cells And

Published OnlineFirst December 2, 2020; DOI: 10.1158/0008-5472.CAN-20-1818

CANCER RESEARCH | TUMOR BIOLOGY AND IMMUNOLOGY

Cyclophosphamide and Vinorelbine Activate Stem-Like CD8þ T Cells and Improve Anti-PD-1 Efﬁcacy in Triple-Negative Breast Cancer A C Paolo Falvo1, Stefania Orecchioni1, Roman Hillje2, Alessandro Raveane1, Patrizia Mancuso1, Chiara Camisaschi1, Lucilla Luzi2, PierGiuseppe Pelicci2, and Francesco Bertolini1

ABSTRACT ◥ Checkpoint inhibitors (CI) instigate anticancer immunity in change resulting from exposure to a mitogen, ligand, or antigen for many neoplastic diseases, albeit only in a fraction of patients. The which it is speciﬁc, as well as APC-to-T-cell adhesion. This tran- þ clinical success of cyclophosphamide (C)-based haploidentical scriptional program also increased intratumoral Tcf1 stem-like þ stem-cell transplants indicates that this drug may re-orchestrate CD8 T cells and altered the balance between terminally and the immune system. Using models of triple-negative breast cancer progenitor-exhausted T cells favoring the latter. Overall, our data (TNBC) with different intratumoral immune contexture, we dem- support the clinical investigation of this therapy in TNBC. onstrate that a combinatorial therapy of intermittent C, CI, and vinorelbine activates antigen-presenting cells (APC), and abrogates Signiﬁcance: A combinatorial therapy in mouse models of breast local and metastatic tumor growth by a T-cell–related effect. Single- cancer increases checkpoint inhibition by activating antigen- þ þ cell transcriptome analysis of >50,000 intratumoral immune cells presenting cells, enhancing intratumoral Tcf1 stem-like CD8 þ after therapy treatment showed a gene signature suggestive of a T cells, and increasing progenitor exhausted CD8 T cells.

Introduction TNBC (4T1 and EMT6) to investigate whether C and V, in different schedules and doses, could be more effective than T, D, or P. Checkpoint inhibitors (CI) have shown an unprecedented clinical The first series of preclinical studies reported here have clearly activity in several types of cancer, but only a fraction of patients has indicated that intermittent, medium-dosage C (140 mg/kg, C140), a benefit from CIs administered as a single therapy. Moreover, in some dosage already known to have potent effects on the immune sys- patients, the clinical responses are limited in time (1). These observa- tem (11), was significantly more effective than lower, continuous doses tions are promoting preclinical and clinical studies to improve CI of the same drug (20 mg/kg, C20), and that intermittent C140, in efficacy by means of combinatorial therapies (2). association with V and CIs, were significantly more effective than The clinical success of cyclophosphamide (C)-based haploiden- combinatorial regimens of CIs plus T, D, or P. Thus, we investigated by tical stem cell transplants in hematologic malignancies indicates means of neutralizing mAbs, single-cell transcriptome analysis, and that this drug has the potential to re-orchestrate the immune system flow cytometry what immune cells were involved in these anti-TNBC against cancer cells (3). Along a similar way, C is administered mechanisms and how these drugs reshaped the circulating and intra- before CAR-T cell infusion to improve their clinical efficacy (4). tumoral immune cell landscape in cancer-bearing mice. We have previously found that low-dose, daily C, in association A recent meta-analysis from clinical trials involving anti-PD-1 and with vinorelbine (V) was able to improve the preclinical efficacy of anti-PD-L1 CIs indicated that anti-PD-1 had a favorable survival CIs anti-PD-1 and anti-PD-L1 in models of breast cancer and outcomes and a safety profile comparable to that of anti-PD- lymphoma (5). As randomized clinical trials have recently indicated L1 (12). For this reason, we are showing here only the results obtained that the addition of CIs to other chemotherapy drugs such as in our clinical models with the anti-PD-1 CI. At the present time, we taxanes (T), doxorubicin (D), or platinum (P) might be beneficial have no evidence that anti-PD-L1 can be more effective of anti-PD-1 in in patients with triple-negative breast cancer (TNBC; refs. 6–10), we these models. designed this study in two preclinical, immunocompetent models of

1Laboratory of Hematology-Oncology, European Institute of Oncology IRCCS, Materials and Methods Milan, Italy. 2Department of Experimental Oncology, European Institute of Cell culture Oncology IRCCS, Milan, Italy. The 4T1 and the EMT6 TNBC cell lines were purchased from Note: Supplementary data for this article are available at Cancer Research ATCC, expanded and stored according to the producer's instructions. Online (http://cancerres.aacrjournals.org/). Cells were tested and authenticated by the StemElite ID System P. Falvo and S. Orecchioni contributed equally to this article. (Promega). Cells were tested every 6 months for Mycoplasma by Corrected online June 2, 2021. means of the ATCC Universal Mycoplasma Detection Kit 30-1012, cultured for no more than 2 weeks and used for no longer than Corresponding Author: Francesco Bertolini, European Institute of Oncology, via Ripamonti 435, Milan 20141, Italy. Phone: 39-02-574-89535; Fax: 39-02-574- 15 passages. 89537; E-mail: [email protected] Cell line transduction Cancer Res 2021;81:685–97 The 4T1 and EMT6 cell lines were stably transduced with lentiviral doi: 10.1158/0008-5472.CAN-20-1818 vector expressing stably luciferase under the control of CMV promot- 2020 American Association for Cancer Research. er: pLenti CMV Puro LUC (w168-1; Addgene plasmid, #17477),

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carrying puromycin resistance as selective marker. To infect 4T1 and in drinking water (C20) and 140 mg/kg (C140) in intraperitoneal EMT6 cell lines, 293T cells were cotransfected with 10 mg of lentiviral injection as already shown in refs. 5 and 11. V was used at 9 mg/kg as we vector, 3 mg PMD2G envelope plasmid, 2.5 mg of REV packaging have previously shown in ref. 5. P was used at 0.25 mg/kg (15), T was plasmid, and 5 mg of RRE transfer plasmid using calcium phosphate used at 20 mg/kg (16), D was used at 2.9 mg/kg (15). Chemothera- transfection system. After 16 hours, media was removed and replaced peutic drugs were dissolved in PBS and administrated, once a week, with 5 mL of fresh medium. Viral supernatant was then collected at every 6 days, for 3 weeks by intraperitoneal injection. Mouse mono- 24 and 48 hours and added directly to 4T1 and EMT6 cell lines plated clonal PD-1-targeting antibody was purchased by Bioxcell (clone at 1 105 supplemented with 8 mg/mL of polybrene (Merck). Infection RMP1-14) and was administered intraperitoneally 0.2 mg/mouse was performed for 3 hours at 37C twice in a day for two separate every 2 days for a total of five doses. Monoclonal neutralizing days. After 48 hours from first cycle of infection, infected cells were antibodies were purchased by Bioxcell. Anti-CD4 (clone GK1.5), selected adding 1.5 mg/mL (for 4T1 cell line) and 2 mg/mL (for EMT6 anti-CD8a (clone 2.43), anti-CD19 (clone 1D3), anti-CD11b (clone cell line) puromycin (Merck) to the medium. Selection was carried out M1/70), anti-CD103 (clone M290), and anti-CD122 (clone TM-beta1) for 72 hours, until not infected control cells were dead. were administrated 300 mg/mouse twice a week for a total of five doses. In vivo studies Experiments involving animals were approved by the Italian Tumor dissociation and cell sorting for scRNA-seq library Ministry of Health and have been done in accordance with the preparation applicable Italian laws (D.L.vo 26/14 and following amendments), Mice tumors were surgically removed after 28 days from tumor the Institutional Animal Care and Use Committee, and the institu- injection to generate a single cell suspension. Briefly, after mechanical tional guidelines at the European Institute of Oncology. In vivo studies dissociation, tumors were placed in culture medium (1:1 of DMEM were carried out in 6 to 8 weeks old immune-competent BALB/ with high glucose and Ham's F-12 Nutrient Mixture; EuroClone) cOlaHsd female mice (Envigo) in mouse facilities at the European supplemented by 2 mg/mL collagenase (Merck) and 0.1 mg/mL Dnase Institute of Oncology–Italian Foundation for Cancer Research (FIRC) I (Qiagen), and digested for 1 to 2 hours at 37C. A single cell Institute of Molecular Oncology (IEO–IFOM, Milan, Italy) campus. suspension was obtained by sequential dissociation of the fragments To generate syngeneic models of TNBC in BALB/c, 2 105 4T1-LUC by gentle pipetting, to further disintegrate cell clumps, followed by and 5 105 EMT6-LUC were injected in the mammary fat pad filtration through a 100-mmol/L nylon mesh. After cell suspension as we described previously (5). Tumor growth was monitored preparation, cells were incubated with PE-conjugated murine CD45 weekly using In Vivo Imaging System (IVIS; PerkinElmer). Briefly, antibody (BD Biosciences, clone 30-F11) for 20 minutes at 4C. mice were intraperitoneally injected with 150 mg/kg of XenoLight Cells were then washed twice with PBSþ0.2% BSA and prepared D-Luciferin–Kþ Salt Bioluminescent Substrate (PerkinElmer). After for cell sorting. Before sorting, cells were costained with 0.1 mg/mL þ 10 minutes, animals were anaesthetized with isofluorane apparatus 40,6-diamidino-2-phenylindole (DAPI, Merck). CD45 DAPI cells and images acquired using Living Image Software (PerkinElmer; were sorted using FACS Fusion sorter (BD Biosciences). Post-sorting Supplementary Figs. S1A and S1B). Tumor dimension was finally asses- cell purity was assessed by FACS analysis (Supplementary Figs. S2A sed using ImageJ software, by converting pixel intensity into centi- and S2B) and purity was assessed to be higher than 90%. Cells were meters and determining finally tumor mass sphere volume in mm3. then counted and 5,000 cells/conditions underwent scRNA-seq library preparation following 10X Genomic V2 and V3 protocol. TNBC metastatic models TNBC resection was performed 28 (for all EMT6 experimental Tumor dissociation for FACS analysis groups and for 4T1 control, anti-PD-1, Dþ anti-PD-1, Tþ anti-PD-1, Mice tumors were surgically removed and processed to obtain cell Pþ anti-PD-1 experimental groups) or 70 (for C140-treated experi- suspension as explained in the previous paragraph. Gating strategies mental 4T1 tumor) days after tumor implant as we described previ- are summarized in Supplementary Figs. S3A and S3B. Briefly, viable ously (5, 13, 14). Fifteen days after mastectomy, mice were sacrificed by cells (negative for 7-aminoactinomycin, 7AAD; Beckman Coulter) carbon dioxide inhalation and lung tissues were removed. To confirm were stained with antibodies cocktail mix containing anti-CD45, anti- þ þ þ þ the presence of metastases, sections were cut and stained with hema- CD3e, anti-CD8, anti-CD4 to identify CD3 CD8 and CD3 CD4 T toxylin and eosin (H&E), as described previously (5). In brief, lungs cells. Than cells were stained with anti-CD279 (anti-PD-1) and were fixed in 4% phosphate-buffered formalin and embedded in anti-Tim3 antibodies. Percentage was referred to alive lymphocytes. paraffin. Five micrometers thick sections of lungs were made, and slides were counterstained with H&E for the detection of metastases. Alignment and quality control analyses Images were acquired with a ScanScope XT scanner (Leica) and After demultiplexing, reads were aligned with Cell Ranger v3.0.2 analyzed with Aperio Digital Pathology software (Leica). To assess to the mm10 reference genome using the GENCODE vM16 feature the metastases in the lungs, ImageJ software was used. Areas occupied annotation (17). Transcript counts from genes with the same symbol by the metastases was divided to total lungs' volume to assess the but different ENSEMBL ID were summed up, resulting in a total of percentage of metastasis in the lungs. 53,278 unique genes. We merged the raw transcript counts from all six treatments for In vivo therapy each cell line using Seurat v3 (18, 19). Then, cells with 500 transcripts Tumor-bearing mice (n ¼ 5 per study arm) were treated with either or less, 50,000 transcripts or more, fewer than 250 expressed genes, vehicle or with different drugs used as single agents or in combination. fewer than 0.80 log10 genes per UMI (Supplementary Figs. S4A and Drug dosages used in this study are the gold-standard chemothera- S4B), or more than 15% mitochondrial reads were removed from the peutic drugs for TNBC and doses were based on literature data analysis (Supplementary Figs. S5A and S5B; Supplementary Tables S1 associated with no or acceptable toxicity, as well as no significant and S2). Similarly, genes expressed in fewer than 5 cells were removed changes in mouse weight. C was used at two different doses: 20 mg/kg from the analysis, resulting in 22,806 and 23,757 genes on 4T1 and

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Cyclophosphamide and Vinorelbine Activate Stem-Like T Cells

EMT6 cell line, respectively (Supplementary Figs. S6A and S6B). Gene expression evaluation Downstream analyses, if not specifically mentioned otherwise, were Expression values of single genes shown in violin plots represent the carried out using Seurat v3. log-normalized counts from “RNA” assay. To represent the expression of gene sets, we either calculated a module score with the “AddModule- Normalization, batch correction, clustering, and cell-cycle Score()” function, or computed the average expression with the analysis “AverageExpression()” function applied to the “SCT” assay. Transcript counts were log-normalized using the “NormalizeData ()” function. Canonical correlation analysis (CCA) was used to inte- Gene set variation analysis grate data generated with the v2 chemistry and the v3 chemistry of the To perform the gene set variation analysis (GSVA), we downloaded 0 “ ” 10 Genomics Single Cell 3 , respectively. First, raw transcript counts the 50 hallmark gene sets (27) using msigdbr package (28). From fi were normalized with the “SCTransform()” function separately for those, we selected 26 pathways of interest with a speci c interest for the each data set of the two chemistry version. Then, the two data sets were proliferation and immune cells activation groups. Then, we removed integrated using the “SelectIntegrationFeatures()” (to select 3000 duplicated genes within and between pathways following the proce- features for integration in each data set), “PrepSCTIntegration(),” dure by Lambrechts and colleagues (29). Finally, for a subset of the data “FindIntegrationAnchors(),” and “IntegrateData().” Subsequently, set containing only T cells, we estimated the GSVA score on the raw fi t principal component analysis (PCA) was performed on the integrated transcript counts, t a linear model and estimated the moderate data set. The first 30 principal components were used to construct a statistic in default mode for each combination of treatments. Shared Nearest Neighbor (SNN) graph, from which clusters were Flow cytometry calculated through the “FindClusters()” function and the resolution At least 100,000 cells per sample were acquired using a 3-laser, parameter set to “0.8.” The Uniform Manifold Approximation and 10-color flow cytometer (Navios; Beckman Coulter). Viable cells Projection (UMAP) method was used for dimensionality reduction (negative for 7-aminoactinomycin, 7AAD; Beckman Coulter) were and calculated from the first 30 principal components. Cell-cycle labelled with a panel of antibodies to analyze immune cell popu- analysis was performed using the "CellCycleScoring()” function and lations. Antibodies list and gating strategies are summarized in the S- and G2M-specific genes downloaded from ensemldb package the Supplementary Table S4. Lymphocytes and myeloid cells were (Supplementary Figs. S7A and S7B; ref. 20). characterized using state-of-the-art markers. Specifically, cells were gated for size, singlets, and then by positive and negative markers: Cell-type assignment þ þ þ þ þ þ CD3 CD4 and CD3 CD8 T cells, CD335 NKs, CD19 Bcells, Cell type annotation was carried out with the SingleR R package þ þ þ þ Gr1-CD11b CD11c monocytes. SSChighCD11b Gr1 granulocytes (21) and the “ImmGenData” reference (22), containing 253 fine þ and CD11c CD11b Gr1 antigen-presenting cells (APC). APC were labels generated from 830 microarray samples of sorted cell popu- also marked with CD80 to determine their activation. For each immune lations. We performed an initial assignment to the finest level cell subtype, we calculated cell absolute value by multiplying the per- possible using the fine labels and then manually grouped together centage to the total number of white blood cells/mL. the cell populations to reach a reasonable granularity that helps exploration of the dataset. The conversion of the manually assigned T-cell receptor analysis groups of cells can be found in Supplementary Table S2. At both T-cell receptor (TCR) clonality was analyzed in the tumor infiltrate levels, cells belonging to labels with less than 20 cells were assigned using FITC Hamster Anti-Mouse Beta TCR Kit (BD Biosciences, clone “ ” to the group Others (Supplementary Table S2). Chi-square sta- H57-597), following manufacturer's protocol. Briefly, tumor was tistics using as expected the T-cell population frequencies of the dissociated as shown in the previous paragraph and 5 105 cells control was performed, and its strength was tested with the Cramer were stained with antibody cocktail mix containing: 7AAD (Beckman V test. Chisq.test() and an in-house R functions were used to Coulter), CD45 APC-Cy7 (BD Biosciences, clone 30 F-11), CD3 APC calculate the two tests respectively (Supplementary Table S3). We (BD Biosciences, clone 145-2C11), CD4 PE (BD Biosciences, clone measured the fold change of the T-cell populations in each treat- GK1.5), CD8 PE-Cy7(BD Biosciences, clone 53-6.7), and a specific F F F ment as result of the expression: i/ c,inwhich i is the frequencies region of VDJ beta chain rearrangment. Viable cells were gated for fi F of the T-cell population in the speci ctreatmentwhilethe c is the CD45/CD3 positivity. Then, cells were gated for CD4 and CD8 frequencies in the control. Correlations between the T-cell fold positivity, and among them we evaluated for each b chain rearrange- changes were performed either with Spearman or Pearson tests ment the percentage of positivity. Finally, fold increase was determined fi according to their normality as in ref. 23. Only the signi cative by dividing the percentage of positivity in the untreated control versus comparisons were plotted. each experimental condition.

Marker genes detection and pathway enrichment analysis Statistical analysis and graphical representation Marker genes were identiﬁed through the “FindAllMarkers()” Data were expressed as means SEM (in case of normal distri- Seurat function with a Wilcoxon test. Parameters were set to only bution). Normal distribution was assessed using Shapiro Wilk's nor- identify overexpressed genes that are expressed in at least 70% of the mality test. To compare two sample groups, either the Student t test or cells of the cell population of interest, for example, cluster, with a log the Mann–Whitney U test was used on the basis of normal or not fold-change of at least 0.25 compared with all other cells. Furthermore, normal distribution. Statistical analysis was carried out Prism 8.3.1. only genes that passed statistical testing with a P value of 0.01 or (GraphPad) and R (URL http://www.R-project.org; ref. 30). Graphical lower were retained. Pathways enriched in the marker genes were model was created using Biorender Software (https://biorender.com/). retrieved using the “getEnrichedPathways()” function from the cere- broApp R package (24), which queries the Enrichr database (25, 26). Data availability Results were ﬁltered for pathways with an adjusted P value of 0.05 Single-cell transcriptome data of tumor samples and codes used for or lower. their analysis are available from the corresponding author upon request.

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Results previously (5), flow cytometry and single-cell transcriptome analyses fi fi Intermittent, medium-dosage C140, was more effective than con rmed that V-containing regimens were associated with signi cant APC activation, measured by CD80 expression (Fig. 2A and B; other combinatorial regimens including CIs plus low-dose C, or fl regular-dose T, D, or P. The association of CIs and V further Supplementary Fig. S11). Moreover, ow cytometric immunopheno- typic assessment of TCR-V(b) repertoire within tumors suggested a increased the preclinical efficacy of C140 þ þ þ þ significantly larger circulating CD3 CD4 and CD3 CD8 T cells We injected five mice per experimental group with 4T1 and EMT6 clonal expansion in mice treated with C140 when compared with mice cells and treated them with chemotherapeutic agents, CI and chemo- treated with other drugs (Fig. 2C–F). therapeutic agents combined to CI. Chemotherapeutic agents were Using scRNA-seq analyses, we observed that 4T1 and ETM6 administered every 6 days, whereas anti-PD-1 every 2 days for a total of TNBC tumors were significantly different in the landscape of five injections, as shown in the therapeutic scheme presented in intratumoral immune cells: 4T1 tumors were more infiltrated by Supplementary Fig. S8A. We measured tumor growth in both models, lymphoid cells, whereas ETM6 tumors by myeloid cells (Fig. 3A and as shown in Fig. 1A and B and Supplementary Figs. S8B and S8C. and B; Supplementary Table S5). In both models, single-cell tran- In vivo studies in local and metastatic models using both 4T1 and scriptome analyses of more than 50,000 intratumoral immune cells EMT6 TNBC cells clearly indicated that intermittent (i.e., every 6 days), indicated that C140, V, and CI treatment was associated with the C140 was more effective than any other combinatorial regimens largest T-cell intratumoral population, with a predominant pre- including CIs plus daily C20, or T, D, or P at their regular preclinical þ þ sence of CD3 CD4 T helper cells (T 1 cells), known to promote dosages. H antitumor immunity (Fig. 3A and B;ref.32).Toassesscell For the EMT6 model, the triple therapy abrogated tumor growth in diversity, chi-square statistics and Cramer V test were applied for 28 days (P < 0.05), whereas for 4T1-bearing mice, C140 alone both the cell lines using as expected values the T cells frequencies of abrogated tumor growth up to day 28. Therefore, for 4T1 model, the control. The number of cells resulted significative different we prolonged the time schedule to 70 days and we observed that from the control for each treatment in both the cell lines (Supple- the association with V and CIs (anti-PD-1) further increased the mentary Table S5). Moreover, we measured the increase and the preclinical efficacy of C140 (P < 0.001). Tumor resection was per- fold change of the T-cell populations for each treatment compared formed for 4T1 tumor on day 28 for control, anti-PD-1, Dþ anti-PD-1, with the control. We identified a strong correlation between the Tþ anti-PD-1, and Pþ anti-PD-1, and on day 70 for all C-treated increase of T cells for PD1, Cisplatin þ PD1 and triple treatments groups; whereas for EMT-6 tumor, resection was performed at day 28 between the two lineages (Supplementary Figs. S12A–S12F). for all treatment groups. After tumor resection, we observed that the triple therapy abrogated metastatic tumor dissemination in the lungs Single-cell transcriptome analyses showed several unique in the 4T1 model (P < 0.001; Fig. 1C). In the EMT6 model, we observed T-cell activation patterns associated with C140, V, and CI a reduction in metastases with the triple therapy compared with treatment controls, even though this mouse model is known to generate less As intratumoral T cells were more abundant in mice treated with metastases (Fig. 1C and D; Supplementary Figs. S8D and S8E; ref. 31). C140, V, and CIs, we tested the hypothesis of a phenotypic change in these T cells and performed an analysis of hallmark pathway gene T cells are needed to control tumor growth in mice treated signatures as in ref. 28. We compared the pathways observed in control with C140, V, and CIs and after C140, V, and CI treatment and observed that this treatment To further demonstrate that immunity plays a crucial role in increased the expression of pathways involving proliferation (i.e., tumor eradication, we performed preclinical studies with neutralizing – myc target, G2 M checkpoint) and T-cell activation (i.e., IFNa and monoclonal antibodies targeting in separate experiments T, B, NK, and g response, Fig. 4A and B). Our data strongly suggest that C140, V, and myeloid cells as shown in the therapeutic scheme presented in CI induce T-cell proliferation and activation. fi Supplementary Fig. S9A. Neutralizing antibody ef cacy was tested Moreover, we studied the expression of T-cell different activating by FACS as shown in the Supplementary Figs. S9B to S9D. In TNBC- genes (Cd27, Cd28, Cd69, Cd2, and IFNg among others, see Fig. 4C bearing mice treated with C140, V, and CIs, the combinatorial therapy fi þ þ and D), and con rmed that after C140, V, and CI these genes were demonstrated that CD3 CD8 (in 4T1-bearing mice), and þ þ þ þ more expressed in intratumoral T cells, strongly suggesting that the CD3 CD8 plus CD3 CD4 T cells (in ETM6-bearing mice) were therapy may increase T-cell activation against the tumor. needed to abrogate TNBC growth (P < 0.05; Fig. 1E and F). þ Finally, because we have noticed that T-cell activation pathway To a much lesser extent, in ETM6-bearing mice also CD11b seems to be upregulated by triple therapy, we investigated which genes P þ P þ myeloid cells ( < 0.01), CD19 B cells ( < 0.01), and CD122 NK are mostly expressed. For both models, we ended up with a list of P cells ( < 0.01) had a role in reducing TNBC growth. 20 genes comprising genes such as Cd3d, Ptprc, Cd69, Lat, Lck, Cd2, and Cd3e among others, suggestive of a change in morphology Chemotherapy induces selective modulation of circulating and and behavior resulting from exposure to a mitogen, cytokine, chemo- intratumoral immune cell subsets and circulating T-cell clones kine, cellular ligand, or an antigen for which it is specific, as well as As we previously observed (5), during therapy the circulating APC-to-T-cell adhesion (Fig. 5A and B). landscape and the intratumoral contexture of immune cells were To further corroborate this hypothesis, we observed which genes are markedly different. Therefore, we decided to analyze the immune cell mostly modulated by the therapy. We observed that for 4T1 model landscape in the peripheral blood by flow cytometry, as shown in 7 genes are differentially expressed (Fig. 5C), whereas for EMT6 model Supplementary Figs. S10A and S10B, and within the tumor by single- 11 genes (Fig. 5D). EnrichR was then used to test for enrichment of cell RNA seq analysis (scRNA-seq). these genes for specific signaling pathways, based on annotations from In the peripheral blood, C140-containing regimens significantly the KEGG database, demonstrating that they modulated pathways reduced the number of circulating lymphoid and myeloid cells in both involved in antigen procession and presentation for both models (P < mouse models (Supplementary Figs. S10A and S10B). As also reported 0.05; Fig. 5E and F).

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Days after tumor injection Days after tumor injection CD

Days after tumor injection Days after tumor injection

Figure 1. The triple therapy C140þVþanti-PD1 is highly effective in two mouse models of TNBC and relies on T cells. A, 4T1 tumor growth curve of mice treated with chemotherapeutic agents, CI, or chemotherapeutic agents combined with CI. Dashed line indicates the mastectomy. B, EMT6 tumor growth curve of mice treated with chemotherapeutic agents, CI, or chemotherapeutic agents combined with CI. The experimental groups are represented in the same way shown in A. Dashed line indicates the mastectomy. C, 4T1 quantification of lung metastases in the experimental conditions described in A. Metastases percentage was obtained dividing the area occupied by metastases in the lungs over the whole lung areas. D, EMT6 quantification of lung metastases in the experimental conditions described in A. Metastases percentage was obtained dividing the area occupied by metastases in the lungs over the whole lung areas. E, 4T1 tumor growth curve of mice treated with triple therapy (C140þVþanti-PD1) and different neutralizing mAbs targeting different component of immune cells: CD4þ helper T cells (anti-CD4), CD8þ cytotoxic T cells (anti-CD8), macrophage (anti-CD11b), B cells (anti-CD19), dendritic cells (anti-CD103), and NK (anti-CD122). F, EMT6 tumor growth curve of mice treated with identical experimental condition shown in E (n ¼ 5 per study arm); Shapiro–Wilk test was applied to define normal distribution. Distribution is normal in graphs A, B, C, and F, whereas it is not normal in the graph E. Therefore, two-tail Mann–Whitney was applied to E, and Student t test was applied to A, B, C, and F. Statistical analysis was done for each condition compared with C140þVþanti-PD1 for graphs A, B, E,andF, whereas it was done for C140þVþanti-PD1 compared with control for the graph E. , P < 0.05; , P < 0.01; , P < 0.001.

The C140, V, and CI therapy moves the balance of progenitors exhausted T cells, which may have beneficial antitumoral effects in exhausted versus terminal exhausted T cells patients with melanoma (33). Progenitors exhausted T cells are We asked what was PD-1 contribution to the treatment. It is defined to be Tim3 (gene name Havcr2), whereas terminally widely known that anti-PD-1 favors the selection of progenitors exhausted T cells, which have poor anticancer function, are defined

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ABFigure 2. V induces APC activation, whereas C140 activates T-cell clonality. A, FACS analysis of CD80þ dendritic cells (green) over total APC (light blue) in mice treated with chemotherapeutic agents, CI, or chemotherapeutic agents combined with CI injected with 4T1 cells after 3 weeks of treatment. B, FACS analysis of CD80þ dendritic cells as shown in A in mice injected with EMT6 cells after 3 weeks of treatment. C, CD3þCD4þ TCR clonality in 4T1 tumors. On x-axis, 15 com- mon Vb chain recombinations are present, whereas on y-axis the fold change increase is compared with CDuntreated mice. D, CD3þCD8þ TCR clonality in 4T1 tumor, as shown in C. E, CD3þCD4þ TCR clonality in EMT6 tumor, as shown in C. F, CD3þCD8þ TCR clonality in EMT6 tumor as shown in D (n ¼ 5perstudyarm);Shapiro–Wilk test was applied to deﬁne normal distribution. Distribution is normal in graphs A and B,andStudent t test was applied to A and B. Statistical analysis was done for each condition compared with each control. , P < 0.05. N EF

þ þ as Tim3 . Therefore, we wondered whether it was the case in our Consistent with this observation, Tim3 T cells, previously model systems. We checked first by scRNAseq whether this treat- designated as terminally exhausted cells, were found predominantly þ ment increased the frequency of intratumoral Tcf1 stem-like in control and chemo-alone groups, whereas the addition of CI to þ CD8 T cells (P < 0.001; Fig. 6A). Synergy between C140/V and chemotherapy significantly decreased these cells. To further cor- CI might be due, at least in part, to CI selective activity on roborate this hypothesis, we also validated the presence of Entpd1, þ progenitor exhausted CD8 tumor-infiltrating lymphocyte (TIL), another marker on terminally exhausted T cell. Consistent with which can respond to anti-PD-1 and were significantly increased in Tim3, also Entpd1 is less expressed in triple treatment, suggesting mice treated with chemotherapy and CI when compared with C140 a selection towards progenitors exhausted T cells in presence of and C140/V treatments (P < 0.001; Fig. 6B for both 4T1 and EMT6 anti-PD-1 (P < 0.001; Fig. 7). models). To further corroborate the scRNAseq data, we performed ex vivo validation of our data by FACS analysis. Briefly, after tumor injection and treatments, tumors were digested and co-expression Discussion þ of PD-1 and Tim-3 was assessed in CD8 T cells. Gating strategies TNBC is among the most aggressive and lethal types of breast are shown in Supplementary Figs. S3A and S3B. Upon chemother- cancer, and currently available therapies have an unsatisfactory impact apy and anti-PD1 treatments, co-expression of PD-1 and Tim3 on patients' survival. The association of CIs with chemotherapy has decreased only in chemotherapy-treated mice. This decrease was shown some encouraging results in randomized clinical trials enrolling more evident when anti-PD1 was present in synergy with both C patients with TNBC, but not all the studies have met their end points and V (P < 0.05 for EMT6 and P < 0.01 for 4T1 model; Fig. 6C). and there has been no clear evidence of what should be considered the

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Cyclophosphamide and Vinorelbine Activate Stem-Like T Cells

Figure 3. Immune cells landscape in 4T1 and EMT6 tumors by scRNA-seq analysis. A, Immune cell landscape analyzed by scRNA-seq in 4T1 tumor in the different experimental conditions. The top panel shows the uniform manifold approximation and projection (UMAP), with cells colored by the immune cell type they were assigned to, whereas the bar graph in the bottom panel represents the different immune cell abundance within each treatment. B, Immune cell landscape analyzed by scRNA-seq in EMT6 tumor in the different experimental conditions. The top panel shows the UMAP with cells colored by the immune cell type they were assigned to, whereas the bar graph in the bottom panel represents the different immune cell abundancy within each treatment.

best chemotherapy backbone to be associated with Cis (6–10). We innate immune cell effector functions are enhanced by C in path- þ designed this study to define, at the preclinical level, what could be ways also involving CD11b cells as in the ETM6 model presented considered the most promising combinatorial regimen of chemother- here. Present data clearly indicated that intermittent, medium apy plus CI. We investigated different options including P, D, T, V, and dosage C (C140), in combination with V, was more effective of all C, that is the most effective chemotherapy drugs for this disease. other tested combinations. Interestingly, this triple therapy was Capecitabine (or 5-FU) was not considered as our previous data similarly effective in two models of TNBC that seem to elicit indicated that C and V were more effective when used in combination different immunologic responses, as in 4T1 tumors the immune with Cis (5). cell infiltrate was mainly lymphoid, whereas in ETM6 tumors the C is known to elicit potent effects over the immune system infiltrate included in a larger part myeloid (as well as B) cells. In this including Treg reduction, as wellasNK,MDSCs,andDCexpansion context, it is noteworthy that myeloid cells, and in particular (reviewed in ref. 34). In fact, the clinical use of C after partially- macrophages, are known to mediate tumor resistance to CIs (37, 38). mismatched hematopoietic stem cell transplant has demonstrated The observed decrease in MHC-II is likely due to the decrease in that this drug effectively re-orchestrate also T cells that are pivotal intratumoral myeloid cells. in anticancer immunity (3). The use of C as an immunomodulatory To define the mechanisms crucial for C140, V, and CIs pre- drug has been reported both in preclinical models and in clinical clinical activity, we first identified what immune cells populations trials (11, 35, 36). In addition to effects on the rewiring of T cells, were needed to control tumor progression. In ETM6-bearing mice

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Figure 4. Triple therapy promotes T-cell activation and proliferation. A and B, Differ- ences in pathway activities scored by GSVA between control and triple treatment (C140þVþanti-PD1; A), and between triple and C140þV treatments for T cell as assigned by singleR (B). t scores from a linear model are shown with the threshold set to 3 (gray pathways). Left, 4T1 T cells; right, EMT6 T cells. C, T-cell activity estimated through module score and calculated for genes Cd27, Cd28, Cd69, Cd25, and IFNg in 4T1 (left) and EMT6 (right) tumor immune cells for the different experimental conditions. D, T-cell activity is shown as in C, but presented as expression. Red line, median value.

with a predominant myeloid and B-cell infiltrate (but not in (and B) cell landscape. However, neutralization studies with mAbs 4T1-bearing mice with a predominant lymphoid infiltrate), neu- against T cells clearly indicated a central, more relevant role for þ þ tralization by mAbs of CD11b myeloid cells, CD19 B cells, and/or these cells in the preclinical activity of the C140, V, and CI therapy. þ þ þ CD122 NK cells slightly reduced the preclinical efficacy of the Neutralization of CD3 CD8 T cells completely abrogated the C140, V, and CI therapy, thus suggesting that these immune cell efficacy of this therapy in 4T1-bearing mice, and neutralization of þ þ þ þ populations might participate in TNBC control by this triple CD3 CD4 or CD3 CD8 T cells completely abrogated the effi- therapy when the immune cell infiltrate had a relevant myeloid cacy in ETM6-bearing mice. These partially diverging results

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Figure 5. Triple therapy regulates different genes involved in antigen presenting and presentation. A, Twenty top expressed genes in 4T1 model by triple therapy in the GO term “T cell activation.” B, Twenty top expressed genes in 4T1 model by triple therapy in the GO term “T cell activation.” C, Expression of differentially expressed genes from GO term “T cell activation” in triple treatment (C140þVþanti-PD1) in 4T1 cells compared with cells from all other treatments combined. D, Expression of differentially expressed genes from GO term “T cell activation” in triple treatment (C140þVþanti-PD1) in EMT6 cells compared with cells from all other treatments combined. E, Enrichr results from “KEGG mouse 2019” database for differentially expressed genes identiﬁed for triple treatment in 4T1 cells show enrichment in “Antigen processing and presentation.” Taken from Ernichr site: https://amp.pharm.mssm.edu/Enrichr/. F, Enrichr results from “KEGG mouse 201900 database for differentially expressed genes identiﬁed for triple treatment in 4T1 cells show enrichment in “Antigen processing and presentation.” Taken from Ernichr site: https://amp.pharm.mssm.edu/Enrichr/

underline the need for investigating different in vivo models considered and investigated in chemotherapy regimens associated including infiltrates with myeloid versus lymphoid prevalence. with CIs. The exact mechanism of APC activation by V and the role The central role of T cells in the preclinical activity of C140, V, and of B cells are currently investigated in-depth in other companion CI therapy prompted us to investigate the impact of these drugs studies in our laboratory. over different populations of T cells and over their interactions with Single-cell transcriptome analysis of more than 50,000 intratu- other players. To this aim, single-cell RNA sequencing provided a moural immune cells indicated that C140, V, and CIs induced a uni- robust tool to investigate how seemingly homogenous cell populations que gene signature suggestive of a behavior associated with exposure may differ in their functional properties when treated with a given to a mitogen, a ligand, or an antigen, as well as APC-to-T-cell adhesion. drug, and how diverse cell atlases mediate the drug activity (39). Further analyses in T cells indicated that this triple therapy increas- þ þ Activation of APCs, their processing of tumor antigens, and APC ed the frequency of intratumoural Tcf1 stem-like CD8 T cells interaction with T cells is mandatory for an efficient control of tumor (45, 46). Recent evidence suggested that a successful CI therapy growth by T cells. In fact, several preclinical studies have demon- should generate new T-cell clones rather than reinvigorate only pre- strated that APCs are major players in the anticancer properties of existing TILs (47–49). In this context, it is noteworthy that C140, Cis (40–43). As we and others (5, 44) have previously shown that V is a when compared with P, T, and D, was the drug more efficient in potent activator of APCs, we believe that this drug should always be generating new circulating T cells clones (see Graphical Abstract).

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Figure 6. Triple therapy selects terminally exhausted T cells. A, Tcf1 expression in 4T1 (left) and in EMT6 (right) tumor immune cells split by treatment is shown as violin plot. B, Ex- pression of Tim3 (Havcr2) in 4T1 (left) and in EMT6 (right) tumor immune cells split by experimental condition is shown as violin plot. C, Intratumoral percentage of PD-1þTIM3þ CD3þCD8þ T cells is shown in the bar graph in 4T1 (left) and in EMT6 model (right). Cells were plotted calculating the percentage of positive cells over the lymphocytes (n ¼ 5 per study arm); Shapiro– Wilk test was applied to deﬁne normal distribution. Distribution is normal in both graphs and Student t test was applied. Sta- tistical was done for each condition compared with control. , P < 0.05; , P < 0.01; , P < 0.001.

We performed a differential gene expression analysis to identi- reported that exhausted T cells exhibit phenotypic and functional fy which genes are consistently up- and downregulated after heterogeneity, which permits to these cells to be separated in thetripletreatmentinTcells.Weidentified 131 significantly progenitor exhausted and terminally exhausted T populations. upregulated and 50 significantly downregulated genes (Supple- The transcription factor T-cell factor 1 (Tcf1), encoded by tcf1,is mentary Table S6). Many of the upregulated genes are predicted/ a key regulator of progenitor exhausted T cells. In contrast, termi- pseudogenes or ribosomal genes, and pathway enrichment analysis nally exhausted cells express high levels of Tim-3/ENTPD1, and for these genes did not return any significant results. The pathway other co-inhibitory receptors (51). Progenitor exhausted T cells þ enrichment analysis for the downregulated genes offered more (Tcf1 Tim3 ENTPD1 ) mediate long-term tumor control and interesting results, which deserve to be further investigated and possess stem-like characteristics allowing them to undergo self- validated in future studies. renewal and exclusively respond to CI therapy. Terminally exhaust- þ þ T-cell exhaustion develops in a progressive manner under con- ed T subsets (Tcf1 Tim3 ENTPD1 ) are differentiated from ditions of prolonged antigen stimulation, leading to a heteroge- progenitor cells by higher cytotoxicity, but they have reduced neous population of exhausted T cells. Recent studies (33, 50, 51) long-term survival and are unable to respond to CI.

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Figure 7. PD-1 in combination with chemotherapy favors progenitors exhausted T cells. Expression of Entpd1 (Entpd1) in 4T1 (left) and in EMT6 (right) tumor immune cells split by experimental condition is shown as violin plot. Two-tail Mann–Whitney was applied. , P < 0.05; , P < 0.001.

Our data suggest that the synergy of CIs to chemotherapy is due review and editing. R. Hillje: Conceptualization, data curation, software, formal þ — — in part to its selective activity on progenitor exhausted CD8 TILs, analysis, validation, investigation, writing original draft, writing review and edit- fi ing. A. Raveane: Software, formal analysis, writing—original draft, writing—review which can respond to CIs and are signi cantly increased in animals — þ and editing. P. Mancuso: Software, formal analysis, validation, writing original treated with chemo immunotherapy compared with C140 and draft, writing—review and editing. C. Camisaschi: Formal analysis, validation, C140 plus V treatments alone (Figs. 6A–C and 7 for both 4T1 writing—original draft. L. Luzi: Software, formal analysis, writing—original draft. þ and EMT6). Consistent with this, Tim-3 T cells, previously P. Pelicci: Software, formal analysis, funding acquisition, validation, writing— designated as terminally exhausted cells, were found predominantly original draft. F. Bertolini: Conceptualization, resources, supervision, funding acqui- — in control and chemo-alone groups, whereas the addition of CIs to sition, validation, writing-original draft, project administration, writing review and chemo leads to a significantly decrease of these cells. The triple editing. combination therapy may alter the balance between the terminally Acknowledgments and progenitor exhausted T cells by favoring the latter and improv- This work was supported by the Italian Association for Cancer Research fi ing preclinical ef cacy. (AIRC) and the Italian Ministry of Health. F. Bertolini is a scholar of the US Taken together, our observations indicated that the C140, V, and CI National Blood Foundation. R. Hillje is a PhD student at the European School of therapy had a unique activity over the circulating and intratumoral Molecular Medicine (SEMM). Authors would like to thank Stefania Roma and immune cell landscape. These drugs showed a strong preclinical Giulia Mitola, and the IEO analysis laboratory, in particular, Marco Picozzi, activity in two TNBC models, one associated with a predominant Giulia Ricci, and Giulia Clericuzio for hematologic analysis, IEO imaging facility, fi in particular Simona Ronzoni and Matteo Gallazzi for sorting experiments, IEO lymphoid intratumoral in ltrated and the other associated with a pharmacy, in particular Dr. Emanuela Omodeo Sale and Martina Milani for giving fi predominant myeloid/B-cell in ltrate. As the dosages of this therapy usthechemotherapeuticdrugs,IEOGenomicUnit,inparticularLucaRotta, can be transferred into the clinic with a schedule that seems feasi- Salvatore Bianchi, Thelma Capra, and Lucia Massimiliano for scRNA seq libraries ble (11), these findings can be used to design novel clinical trials in this preparation, IEO cell culture, in particular Cristina Spinelli, Manuela Moia, and neoplastic disease. Donatella Genovese for cells technical supports. Finally, we are thankful to Federica Pisati for the hematoxylin and eosin lung slides preparation and Molpath Authors’ Disclosures unit, in particular Giovanni Bertolot, Giovanna Jodice, and Francesca Mantovani for ScanScope XT scanner acquisition. R. Hillje reports grants from AIRC (Fondazione AIRC per la Ricerca sul Cancro) during the conduct of the study. No disclosures were reported by the other authors. The costs of publication of this article were defrayed in part by the paymentofpagecharges.Thisarticlemustthereforebeherebymarked Authors’ Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. P. Falvo: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, writing—original draft, project administration, writing—review and editing. S. Orecchioni: Conceptualization, data cura- Received May 29, 2020; revised November 24, 2020; accepted November 25, 2020; tion, formal analysis, validation, investigation, writing—original draft, writing— published first December 2, 2020.

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Correction: Cyclophosphamide and Vinorelbine Activate Stem-Like CD8þ T Cells and Improve Anti-PD-1 Efﬁcacy in Triple-Negative Breast Cancer

In the original version of this article (1), a label for an EMT6 sample in the PþaPD1 treatment group was inadvertently misspelled in the dataset, which resulted in an error in Figs. 3B, S4B, S5B, S6B, S12B and S12E, and Tables S1 and S5. These errors have been corrected in the latest online HTML and PDF versions of the article. The authors regret this error.

Reference 1. Falvo P, Orecchioni S, Hillje R, Raveane A, Mancusco P, Camisaschi C, et al. Cyclophosphamide and þ vinorelbine activate stem-like CD8 T cells and improve anti-PD-1 efﬁcacy in triple-negative breast cancer. Cancer Res 2021;81:685–97.

Published online June 15, 2021. Cancer Res 2021;81:3426 doi: 10.1158/0008-5472.CAN-21-0869 2021 American Association for Cancer Research.

AACRJournals.org | 3426 Published OnlineFirst December 2, 2020; DOI: 10.1158/0008-5472.CAN-20-1818

Cyclophosphamide and Vinorelbine Activate Stem-Like CD8+ T Cells and Improve Anti-PD-1 Efficacy in Triple-Negative Breast Cancer

Paolo Falvo, Stefania Orecchioni, Roman Hillje, et al.

Cancer Res 2021;81:685-697. Published OnlineFirst December 2, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-20-1818

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