S4D and S4E was conducted using the DESeq2 R package. The study developed here is based on samples taken from surgical residues available after histopathologic analyses and not required for diagnosis. Accordingly, we proposed the following nomenclature for these different FAPhi CAF-S1 clusters: cluster 0, ecm-myCAF; cluster 1, detox-iCAF; cluster 2, IL-iCAF; cluster 3, TGFβ-myCAF; cluster 4, wound-myCAF; cluster 5, IFNγ-iCAF; cluster 6, IFNαβ-myCAF; and cluster 7, acto-myCAF. By performing scRNA-seq on more than 19,000 CAF-S1 fibroblasts from patients with breast cancer, we identified eight cellular clusters within the CAF-S1 immunosuppressive subset in human breast cancer. Using CAF-S1 cluster–specific signatures (Supplementary Data S2), we observed that ecm-myCAF, TGFβ-myCAF, and wound-myCAF gene expression was enriched in nonresponders compared with responders, whereas detox-iCAF, IL-iCAF, and IFNγ-iCAF clusters were not (Fig. Place freshly seeded flasks in a 37°C, 5% CO2, incubator for at least 24 to 48 hours before processing the cells further. 5H), thereby suggesting that the content in TGFβ-myCAF increases upon coculture with CD4+ CD25+ T lymphocytes. Bottom, same as top for normal fibroblast signature. Myofibroblasts from clusters 0 and 3, characterized by extracellular matrix proteins and TGFβ signaling, respectively, are indicative of primary resistance to immunotherapies. The enrichment in wound-myCAF was also anticorrelated with CTLA4+ CD8+, TIGIT CD8+, CD244+ CD8+, CD244+ NK (Fig. 1B and C). eISSN: 2159-8290 We confirmed the accumulation of iCAF clusters in TN and myCAF clusters in LumA breast cancer (Supplementary Fig. © Cells (Treg panel and CAF-S1 panel) were acquired using ZE5 cell analyzer (Bio-Rad) and analyzed by FlowJo v10.4.2.
As NFAT and STAT are well-known transcriptional regulators of immune checkpoints in T cells, these data indicate that ecm-myCAF promotes upregulation of immune checkpoints at RNA levels in CD4+ CD25+ T lymphocytes, potentially through the activation of NFAT/STAT signaling pathways. Indeed, ecm-myCAF and TGFβ-myCAF clusters are enriched in tumors with high levels of PD-1+, CTLA4+, and TIGIT+ CD4+ T lymphocytes (themselves enriched in Tregs), and low fraction of CD8+ T lymphocytes. a thing may be said to exist, is probably ~20 microns in diameter,313 In addition, myCAFs from cluster 3 are specified by TGFβ-signaling pathway and were defined as “TGFβ-myCAF.” Importantly, we confirmed the existence of the five most abundant clusters by using flow cytometry in breast cancer. Forty-four breast cancer samples were cut into small fragments and digested in CO2-independent medium (Gibco, #18045-054) supplemented with 5% FBS (PAA, #A11-151), 2 mg/mL collagenase I (Sigma-Aldrich, #C0130), 2 mg/mL hyaluronidase (Sigma-Aldrich, #H3506), and 25 mg/mL DNase I (Roche, #11284932001) for 45 minutes at 37°C with shaking (180 rpm).
In these conditions, cell concentration was checked in control samples and was of 200,000 cells/mL. vary from a low of 10 trillion312,931,2185 Do not shake forcefully to avoid foaming. We have previously demonstrated that the global CAF-S1 subpopulation has no direct effect on CD4+ CD25− T cells but increases the proportion of FOXP3+ Tregs among CD4+ CD25+ T lymphocytes (8, 10). At least 5 × 105 events were recorded. Table 1. 8; Supplementary Fig. All these signatures are given in the second tab of Supplementary Data S2. Given the direct effect of specific CAF-S1 clusters on PD-1 and CTLA4 protein levels on Tregs, we next wondered whether some CAF-S1 clusters could be associated with immunotherapy resistance. I, Same as in A testing formalin-fixed paraffin-embedded (FFPE) samples isolated before anti–PD-1 treatment from a cohort of 79 patients with NSCLC (n = 48 nonresponders and n = 22 responders; Supplementary Table S2, cohort 4). We use cookies to ensure that we give you the best experience on our website.
With infectious virus assays or viral antigen assays, even a negative test result may leave open the possible existence of a latent viral genome. Virginie Mieulet and Josh Waterfall for critical reading of the manuscript, as well as Dr. Emanuela Romano, Dr. Itay Tirosh, and the GDR 3697 Micronit for fruitful discussions. Quality Accreditations. in diameter (but may be up to five times larger in very obese people).935,936 To validate the CAF-S1 clusters identified in the first dataset, another set of data corresponding to 1,582 CAF-S1 fibroblasts after quality control and collected from an additive breast cancer sample was analyzed using the Seurat pipeline. Cardiac fibroblast cell number was quantified by counting the number of tdTomato + or GFP + cells identified by DAPI + nuclei using the ImageJ cell counter plugin and normalized to myocardial area, DAPI area, or DAPI + nuclei as specified in figure legends. concerned with respiration, physical and chemical work, and mitotic activity. Human experimental procedures for analyses of tumor microenvironment by F. Mechta-Grigoriou's lab were approved by the Institutional Review Board and Ethics committee of the Institut Curie Hospital group (approval February 12, 2014) and CNIL (Commission Nationale de l'informatique et des Libertésapproval no.
Also confined to the bloodstream are We observed that CAF-S1 obtained by spreading expressed high levels of myCAF genes, whereas CAF-S1 isolated by sorting exhibited high expression of iCAF genes (Fig. a median ~2.5 x 108 platelets/cm3, ~7.0 x 106/cm3 DAPI−, EPCAM−, CD45−, CD31− cells were separated on four subsets (CAF-S1 to CAF-S4) according to FAP and CD29. 4). P values are from DESeq2 analysis. Analysis of primary tumor samples was performed in accordance with the relevant national law and with recognized ethical guidelines (Declaration of Helsinki) on the protection of people taking part in biomedical research. D, Percentage (%) of the different clusters among CAF-S1 fibroblasts based on FACS data. Fresh human breast cancer primary tumors were collected directly from the operating room after surgical specimen macroscopic examination and selection of areas of interest by a pathologist. Efficacy of immuno-oncology treatment was assessed every 8 to 12 weeks by whole-body CT scan, by a weekly multidisciplinary tumor board, including thoracic specialized radiologists, thoracic oncologists, and pulmonologists, according to RECIST v.1.1 criteria, defining objective responders (OR), patients with stable disease (SD), and patients showing tumor progression (Progr) with 20% or more increase in their tumor volume without any clinical benefit.
P values from Pearson correlation test.
However, if you would like to, you can change your cookie settings at anytime. Because of the low number of TN breast cancers in our dataset, we sought to address this question by taking advantage of The Cancer Genome Atlas (TCGA) database, which contains RNA-sequencing (RNA-seq) data from a high number of patients with LumA and TN breast cancer. Number of the included components (PCs) was assessed using the JackStraw procedure implemented in JackStraw and ScoreJackStraw functions. cDNA libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina, #20020594) followed by sequencing on NovaSeq (Illumina). We observed that the coculture of CD4+ CD25+ T cells significantly increased the expression of the TGFβ-myCAF–specific marker LAMP5 at the surface of ecm-myCAF fibroblasts (Fig.
After a rapid wash in PBS+, the cells were stained with an anti-FAP antibody (1:100, R&D Systems, #MAB3715) or isotype control (1:100, R&D Systems, #MAB002) in PBS+ for 40 minutes at room temperature. In addition, although wound-myCAF showed no real association with FOXP3, detox-iCAF and IL-iCAF clusters were negatively correlated with FOXP3 (Fig. E, Left, correlation matrix between the content in the 5 most abundant CAF-S1 clusters among total CAF (DAPI− EPCAM− CD45− CD31− CD235a−) and among CAF-S1 fibroblasts (FAPhi CD29med). Specifically, CAF-S1 fibroblasts stimulate immunosuppression by increasing attraction, survival, and overall content of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) in the tumor microenvironment (8, 10). Such increased CTLA4 expression by Tregs has been suspected to be the biological basis for the additive effect of anti-CTLA4 antibodies when used in combination with anti–PD-1 checkpoint inhibitors. We thank Drs. These data thus support the development of strategies combining PD-1 and/or CTLA4 blockade with therapies targeting CAF-S1 cluster components to overcome primary resistance to immune checkpoint blockade. S4C), suggesting that ecm-myCAF increased the total protein levels in T cells. 1–3 and Supplementary Figs.
Moreover, in each experiment, properties of spread and sorted cells were compared at the same passages. CAF-S1 from clusters 1 and 2 (detox-iCAF and IL-iCAF) are characterized by detoxification and inflammatory signaling and by response to IL and TNF pathways, respectively.
B–G, Impact of iCAF and myCAF clusters on FOXP3+ CD4+ CD25+ T cells. We dissected the most prominent pathways and gene-specific signatures, characterizing each CAF-S1 cluster as follows: ECM (cluster 0/ecm-myCAF), detoxification (cluster 1/detox-iCAF), response to stimuli (cluster 2/IL-iCAF), TGFβ (cluster 3/TGFβ-myCAF), wound healing (cluster 4/wound-myCAF), IFNγ and cytokines (cluster 5/IFNγ-iCAF), IFNα/β (cluster 6/IFNαβ-iCAF), and acto-myosin (cluster 7/acto-myCAF). Reciprocally, Tregs tend to promote the conversion of CAF-S1 fibroblasts from ecm-myCAF to TGFβ-myCAF. As we showed that CAF-S1 fibroblasts exert immunosuppression in breast and ovarian cancers (8, 10), we next investigated whether this function could be exerted by all CAF-S1 clusters or restricted to specific ones (Fig. ISO 9001
C–G, Same as in B for the following immune checkpoints: PD-1 (C), CTLA4 (D), TIGIT (E), TIM3 (F), and LAG3 (G) in FOXP3+ CD4+ CD25+ Tregs. As the content in ecm-myCAF and TGFβ-myCAF in breast cancer was associated with a CD4+-enriched immunosuppressive microenvironment whereas iCAF clusters were not, we compared the function of these myCAF and iCAF clusters on CD4+ CD25+ T cells using functional assays, as previously performed in refs.
As defined in the aforementioned study, we considered patients as “nonresponders” to anti–PD-1 if they showed progressive disease, and as “responders” in the case of complete or partial response.
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