CDK4/6 inhibitors are approved in combination with hormonotherapy as a first-line intervention against advanced/metastatic hormone receptor (HR)+ HER2- breast cancer, reflecting their ability to extend progression-free survival (PFS) and overall survival (OS) in this patient population.1-3 Nonetheless, >50% of women with HR+ breast cancer receiving CDK4/6 inhibitors ultimately progress and succumb to their disease, owing to hitherto poorly characterized mechanisms of acquired resistance.1-3 While CDK4/6 inhibitors have been conceived to inhibit the proliferation of cancer cells, accumulating preclinical and clinical evidence indicates that they also mediate numerous immunostimulatory effects that may contribute to efficacy.4,5 These observations suggest that hitherto unidentified immunological mechanisms may promote resistance to CDK4/6 inhibitors in patients with HR+HER2- breast cancer.
Methods
We harnessed a unique immunocompetent mouse model that closely recapitulates the immunobiology of human HR+HER2- breast cancer – including a cold tumor microenvironment (TME) coupled to poor sensitivity to PD-1 blockers6 – along with scRNAseq, functional assays and blocking/neutralization experiments to dissect the immunological mechanisms underlying resistance to CDK4/6 inhibitors. The Cancer Genome Atlas (TCGA) was interrogated by in silico analysis. Moreover, immunohistochemistry, multispectral immunofluorescence, and circulating immunophenotyping were performed on samples from 3 independent cohorts of patients with HR+HER2- breast cancer (including longitudinal samples obtained before, during and after CDK4/6 inhibition).
Results
Interleukin 17 (IL17)-producing γδ T cells are recruited to mouse HR+HER2- mammary tumors upon CDK4/6 inhibition through a CCL2-dependent mechanism. In this model, circulating IL17 levels correlate with poor OS, and blocking the γδ TCR, neutralizing IL17 or CCL2 equally improve the therapeutic activity of CDK4/6 inhibitors. Patients from the TCGA with a signature of IL17 signaling have poor OS and signs of immunosuppression in the TME. In diagnostic biopsies from patients with HR+HER2- breast cancer, γδ T cell infiltration correlate with tumor grade, and γδ T cells reside in the proximity of PD-L1+ tumor cells and macrophages. Patients with high activated γδ T cells in the circulation have reduced PFS on CDK4/6 inhibitors as compared to their low counterparts. Circulating CCL2 levels augment during CDK4/6 therapy in progressing patients. Finally, tumor-infiltrating γδ T cells increase as compared to baseline in patients relapsing on CDK4/6 inhibitors.
Conclusions
Our findings prompt the initiation of clinical trials comparing standard-of-care CDK4/6 inhibition plus letrozole vs CDK4/6 inhibition plus letrozole and an IL17 blocker (at least three of which are currently approved for psoriasis treatment) in patients with HR+HER2- breast cancer.
Acknowledgements
This work has been partially sponsored by the 2019 Laura Ziskin Prize in Translational Research (#ZP-6177, PIs: Formenti, McArthur) from the Stand Up to Cancer (SU2C).
References
Im SA, Lu YS, Bardia A, Harbeck N, Colleoni M, Franke F, Chow L, Sohn J, Lee KS, Campos-Gomez S, Villanueva-Vazquez R, Jung KH, Chakravartty A, Hughes G, Gounaris I, Rodriguez-Lorenc K, Taran T, Hurvitz S, Tripathy D. Overall survival with ribociclib plus endocrine therapy in breast cancer. N Engl J Med 2019;381(4):307–316. Turner NC, Slamon DJ, Ro J, Bondarenko I, Im SA, Masuda N, Colleoni M, DeMichele A, Loi S, Verma S, Iwata H, Harbeck N, Loibl S, André F, Puyana Theall K, Huang X, Giorgetti C, Huang Bartlett C, Cristofanilli M. Overall survival with palbociclib and fulvestrant in advanced breast cancer. N Engl J Med 2018;379(20):1926–1936. Slamon DJ, Neven P, Chia S, Fasching PA, De Laurentiis M, Im SA, Petrakova K, Bianchi GV, Esteva FJ, Martín M, Nusch A, Sonke GS, De la Cruz-Merino L, Beck JT, Pivot X, Sondhi M, Wang Y, Chakravartty A, Rodriguez-Lorenc K, Taran T, Jerusalem G. Overall survival ribociclib plus fulvestrant in advanced breast cancer. N Engl J Med 2020;382(6):514–524. Petroni G, Buqué A, Zitvogel L, Kroemer G, Galluzzi L. Immunomodulation by targeted anticancer agents. Cancer Cell 2021;39(3):310–345. Petroni G, Formenti SC, Chen-Kiang S, Galluzzi L. Immunomodulation by anticancer cell cycle inhibitors. Nat Rev Immunol 2020;20(11):669–679. Buqué A, Bloy N, Perez-Lanzón M, Iribarren K, Humeau J, Pol JG, Levesque S, Mondragon L, Yamazaki T, Sato A, Aranda F, Durand S, Boissonnas A, Fucikova J, Senovilla L, Enot D, Hensler M, Kremer M, Stoll G, Hu Y, Massa C, Formenti SC, Seliger B, Elemento O, Spisek R, André F, Zitvogel L, Delaloge S, Kroemer G, Galluzzi L. Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer. Nat Commun 2020;11(1):3819
Ethics Approval
Mouse experiments were approved by WCM IACUC (#2019-0022). All human studies were on retrospective, fully deidentified samples collected upon informed consent at respective Institutions.
Genetic deficiency of dystrophin leads to disability and premature death in Duchenne muscular dystrophy, affecting the heart as well as skeletal muscle. Here we report that cardiosphere-derived cells (CDCs), which are being tested clinically for the treatment of Duchenne cardiomyopathy, improve cardiac and skeletal myopathy in the mdx mouse model of DMD and in human Duchenne cardiomyocytes. Injection of CDCs into the hearts of mdx mice augments cardiac function, ambulatory capacity and survival. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice and in human Duchenne cardiomyocytes. The findings further motivate the testing of CDCs in Duchenne patients, while identifying exosomes as next-generation therapeutic candidates.
Summary Cancer immunotherapy trials have had variable success, prompting a search for biomarkers of response. Tertiary lymphoid structures (TLS) have emerged as prognostic for multiple tumor types. These ectopic immunological bodies resemble secondary lymphoid organs with segregated B and T cell zones, but they are heterogeneous in their organization and cellular composition. These features have consequences in terms of prognostication and disease clearance, so there is interest in what drives TLS heterogeneity and corresponding immunological responses. We applied single RNA molecule resolution imaging to study biopsies from triple negative breast tumors harboring TLS where the biopsies were taken longitudinally, prior to therapy, after pembrolizumab and after pembrolizumab with radiation therapy. We developed a computational framework to identify TLS and tumor beds and to align spatial trajectories between the immune and malignant structures for systematic analyses. We identified two tumor types based on immune infiltration profiles in the tumor bed. Immune “infiltrated” tumors were eliminated after pembrolizumab, while “non-infiltrated” tumors saw gains in effector T cells and myeloid cells after pembrolizumab and were cleared after pembrolizumab with RT. TLS from infiltrated tumors had better separation of B and T cell zones and had higher expression of immunoreactivity gene pathways in most cell types. Further, malignant cell MHC expression was higher in the tumor beds of infiltrated tumors, providing one plausible mechanism for the groupings. In non-infiltrated tumors, classical dendritic cells enter the tumor bed from TLS proximal zones after pembrolizumab and bring transcription of the CXCL9 chemokine, which can recruit T cells and promote T cell effector phenotypes and was higher in infiltrated tumors at baseline.
Hormone receptor+ (HR+) breast cancer (BC) is the most frequent cause of BC-related deaths. CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy (ET) emerged as an effective approach for metastatic HR+ BC. However, >60% women with HR+ BC receiving CDK4/6i+ET ultimately relapse, potentially due to activation of poorly characterized immunosuppressive pathways in the tumor microenvironment (TME).1 Thus, strategies breaking resistance to CDK4/6i+ET in women with HR+ BC are urgently awaited. Radiation therapy (RT) mediates immunostimulatory effects that only partially overlap with those of CDK4/6i+ET,2 standing out as a promising therapeutic partner. Consistent with this notion, we recently demonstrated that RT followed by the CDK4/6i palbociclib + ET (RT-P+ET) enables superior tumor control in various immunocompetent mouse models of HR+ BC.3 These findings have inspired the design of a randomized phase II clinical trial testing P+ET vs. RT-P+ET in patients with oligometastatic HR+ BC (CIMER, NCT04563507). In this context, we set out to dissect the immunological mechanisms underlying sensitivity vs. resistance to treatment in HR+ BC exposed to P+ET vs. RT-P+ET.
Methods
To dissect the impact of these treatments on immune contexture in HR+ BC, we performed single-cell RNAseq on CD45+ cells infiltrating MPA/DMBA (M/D)-driven carcinomas established in immunocompetent mice (a unique model of luminal B BC), coupled to bulk RNAseq, bioinformatic analysis on public patient datasets, functional studies on ex vivo immune cells and efficacy studies.
Results
We observed that (1) RT and P+ET alone mediate partial efficacy correlating with accumulation of immunosuppressive TREG and IL17A-producing γδ T cells, respectively, (2) γδ T cell depletion improves the efficacy of P+ET, (3) RT-P+ET mediates superior (but incomplete) tumor control, which is partially offset by CD4+/CD8+ T cell co-depletion and correlates with limited infiltration by γδ T cells and TREGS, but accumulation of PD-L1 expressing myeloid cells and M2-polarized TREM2+ macrophages, which have been ascribed robust immunosuppressive effects in multiple settings4; and (4) that PD-1 blockage does not ameliorate the therapeutic effects of RT-P+ET (not shown), pointing to TREM2+ macrophages as to the main culprits for resistance in this setting.
Conclusions
Our observations suggest that γδ T cells and TREM2+ macrophages support the resistance of HR+ BC to CDK4/6i and RT-CDK4/6i, and hence constitute potential targets to delay disease progression.
References
Pandey et al. Molecular mechanisms of resistance to CDK4/6 inhibitors in breast cancer: a review. Int J Cancer 2019;145(5):1179–1188. Rodriguez-Ruiz et al. Immunological impact of cell death signaling driven by radiation on the tumor microenvironment. Nat Immunol 2020;21(2):120–134. Petroni et al. Radiotherapy delivered before CDK4/6 inhibitors mediates superior therapeutic effects in ER + Breast cancer. Clin Cancer Res 2021;27(7):1855–1863. Xiong et al. A gene expression signature of TREM2 hi macrophages and γδ T cells predicts immunotherapy response. Nat Commun 2020;11(1):5084.
Ethics Approval
Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Weill Cornell Medical College (n° 2019–2022).
Triple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (∼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB.
Understanding how intratumoral immune populations coordinate antitumor responses after therapy can guide treatment prioritization. We systematically analyzed an established immunotherapy, donor lymphocyte infusion (DLI), by assessing 348,905 single-cell transcriptomes from 74 longitudinal bone marrow samples of 25 patients with relapsed leukemia; a subset was evaluated by both protein- and transcriptome-based spatial analysis. In acute myeloid leukemia (AML) DLI responders, we identified clonally expanded ZNF683 + CD8 + cytotoxic T lymphocytes with in vitro specificity for patient-matched AML. These cells originated primarily from the DLI product and appeared to coordinate antitumor immune responses through interaction with diverse immune cell types within the marrow microenvironment. Nonresponders lacked this cross-talk and had cytotoxic T lymphocytes with elevated TIGIT expression. Our study identifies recipient bone marrow microenvironment differences as a determinant of an effective antileukemia response and opens opportunities to modulate cellular therapy.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, economical and widely used methods for evaluating gene expression. However, the utility of this method continues to be undermined by a number of challenges including normalization using appropriate reference genes. The need to develop tailored and effective strategies is further underscored by the burgeoning field of extracellular vesicle (EV) biology. EVs contain unique signatures of small RNAs including microRNAs (miRs). In this study we develop and validate a comprehensive strategy for identifying highly stable reference genes in a therapeutically relevant cell type, cardiosphere-derived cells. Data were analysed using the four major approaches for reference gene evaluation: NormFinder, GeNorm, BestKeeper and the Delta Ct method. The weighted geometric mean of all of these methods was obtained for the final ranking. Analysis of RNA sequencing identified miR-101-3p, miR-23a-3p and a previously identified EV reference gene, miR-26a-5p. Analysis of a chip-based method (NanoString) identified miR-23a, miR-217 and miR-379 as stable candidates. RT-qPCR validation revealed that the mean of miR-23a-3p, miR-101-3p and miR-26a-5p was the most stable normalization strategy. Here, we demonstrate that a comprehensive approach of a diverse data set of conditions using multiple algorithms reliably identifies stable reference genes which will increase the utility of gene expression evaluation of therapeutically relevant EVs.
Bladder cancer (BLCA) is more common in men but more aggressive in women. Sex-based differences in cancer biology are commonly studied using a murine model with BLCA generated by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). While tumors in the BBN model have been profiled, these profiles provide limited information on the tumor microenvironment. Here, we applied single-cell RNA sequencing to characterize cell-type specific transcriptional differences between male and female BBN-induced tumors. We found proportional and gene expression differences in epithelial and non-epithelial subpopulations between male and female tumors. Expression of several genes predicted sex-specific survival in several human BLCA datasets. We identified novel and clinically relevant sex-specific transcriptional signatures including immune cells in the tumor microenvironment and it validated the relevance of the BBN model for studying sex differences in human BLCA. This work highlights the importance of considering sex as a biological variable in the development of new and accurate cancer markers.
Abstract Molecular barcoding has provided means to link genotype to phenotype, to individuate cells in single-cell analyses, to enable the tracking of evolving lineages, and to facilitate the analysis of complex mixtures containing phenotypically distinct lineages. To date, all existing approaches enable retrospective associations to be made between characteristics and the lineage harbouring them, but provide no path toward isolating or manipulating those lineages within the complex mixture. Here, we describe a strategy for creating functionalized barcodes that enable straightforward manipulation of lineages within complex populations of cells, either marking and retrieval of selected lineages, or modification of their phenotype within the population, including their elimination. These “SmartCodes” rely on a simple CRISPR-based, molecular barcode reader that can switch measurable, or selectable markers, on or off in a binary fashion. While this approach could have broad impact, we envision initial approaches to the study of tumour heterogeneity, focused on issues of tumour progression, metastasis, and drug resistance.
Abstract Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 ( CDH12, N-Cadherin 2 ), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a ( ITGA1 ), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.
