Abstract:Summary
Breast cancer is a heterogeneous disease. Tumor cells and associated healthy cells form ecosystems that determine disease progression and response to therapy. To characterize features of breast cancer ecosystems and their associations with clinical data, we analyzed 144 human breast tumor and 50 non-tumor tissue samples using mass cytometry. The expression of 73 proteins in 26 million cells was evaluated using tumor and immune cell-centric antibody panels. Tumors displayed individuality in t… Show more
“…We identified about 25% of total immune cells infiltrating BCs as CD14 + TAMs that express variable levels of CD163, underlying the phenotypic heterogeneity in TAMs. Through high‐dimensional analysis approaches, several recent reports have uncovered TAM heterogeneity at the single‐cell level in distinct tumor types, including BC, clear cell renal cell carcinoma, melanoma and lung adenocarcinoma . These studies revealed a great variety of distinct phenotypic TAM subsets that co‐exist by sharing the expression of well‐established M1 and M2 markers.…”
Objectives
The accumulation of tumor‐associated macrophages (TAMs) is correlated with poor clinical outcome, but the mechanisms governing their differentiation from circulating monocytes remain unclear in humans.
Methods
Using multicolor flow cytometry, we evaluated TAMs phenotype in 93 breast cancer (BC) patients. Furthermore, monocytes from healthy donors were cultured in the presence of supernatants from dilacerated primary tumors to investigate their differentiation into macrophages (MΦ) in vitro. Additionally, we used transcriptomic analysis to evaluate BC patients’ blood monocytes profiles.
Results
We observed that high intra‐tumor CD163‐expressing TAM density is predictive of reduced survival in BC patients. In vitro, M‐CSF, TGF‐β and VEGF from primary tumor supernatants skewed the differentiation of healthy donor blood monocytes towards CD163highCD86lowIL‐10high M2‐like MΦ that strongly suppressed CD4+ T‐cell expansion via PD‐L1 and IL‐10. In addition, blood monocytes from about 40% of BC patients displayed an altered response to in vitro stimulation, being refractory to type‐1 MΦ (M1‐MΦ) differentiation and secreting higher amounts of immunosuppressive, metastatic‐related and angiogenic cytokines. Aside from showing that monocyte transcriptome is significantly altered by the presence of BC, we also demonstrated an overall metabolic de‐activation in refractory monocytes of BC patients. In contrast, monocytes from sensitive BC patients undergoing normal M1‐MΦ differentiation showed up‐regulation of IFN‐response genes and had no signs of metabolic alteration.
Conclusion
Altogether, our results suggest that systemic factors skew BC patient blood monocytes towards a pro‐metastatic profile, resulting in the accumulation of further polarised CD163high TAMs resembling type‐2 MΦ (M2‐MΦ) in the local BC microenvironment. These data indicate that monitoring circulating monocytes in BC patients may provide an indication of early systemic alterations induced by cancer and, thus, be instrumental in the development of improved personalised immunotherapeutic interventions.
“…We identified about 25% of total immune cells infiltrating BCs as CD14 + TAMs that express variable levels of CD163, underlying the phenotypic heterogeneity in TAMs. Through high‐dimensional analysis approaches, several recent reports have uncovered TAM heterogeneity at the single‐cell level in distinct tumor types, including BC, clear cell renal cell carcinoma, melanoma and lung adenocarcinoma . These studies revealed a great variety of distinct phenotypic TAM subsets that co‐exist by sharing the expression of well‐established M1 and M2 markers.…”
Objectives
The accumulation of tumor‐associated macrophages (TAMs) is correlated with poor clinical outcome, but the mechanisms governing their differentiation from circulating monocytes remain unclear in humans.
Methods
Using multicolor flow cytometry, we evaluated TAMs phenotype in 93 breast cancer (BC) patients. Furthermore, monocytes from healthy donors were cultured in the presence of supernatants from dilacerated primary tumors to investigate their differentiation into macrophages (MΦ) in vitro. Additionally, we used transcriptomic analysis to evaluate BC patients’ blood monocytes profiles.
Results
We observed that high intra‐tumor CD163‐expressing TAM density is predictive of reduced survival in BC patients. In vitro, M‐CSF, TGF‐β and VEGF from primary tumor supernatants skewed the differentiation of healthy donor blood monocytes towards CD163highCD86lowIL‐10high M2‐like MΦ that strongly suppressed CD4+ T‐cell expansion via PD‐L1 and IL‐10. In addition, blood monocytes from about 40% of BC patients displayed an altered response to in vitro stimulation, being refractory to type‐1 MΦ (M1‐MΦ) differentiation and secreting higher amounts of immunosuppressive, metastatic‐related and angiogenic cytokines. Aside from showing that monocyte transcriptome is significantly altered by the presence of BC, we also demonstrated an overall metabolic de‐activation in refractory monocytes of BC patients. In contrast, monocytes from sensitive BC patients undergoing normal M1‐MΦ differentiation showed up‐regulation of IFN‐response genes and had no signs of metabolic alteration.
Conclusion
Altogether, our results suggest that systemic factors skew BC patient blood monocytes towards a pro‐metastatic profile, resulting in the accumulation of further polarised CD163high TAMs resembling type‐2 MΦ (M2‐MΦ) in the local BC microenvironment. These data indicate that monitoring circulating monocytes in BC patients may provide an indication of early systemic alterations induced by cancer and, thus, be instrumental in the development of improved personalised immunotherapeutic interventions.
“…By compiling and then comparing the levels of expression of many proteins across multiple cell types, single-cell mass cytometry is a robust readout to clarify the differentiation of expression patterns in multiple immune cell types. 20,22,23,25 Our comprehensive single-cell analysis of tumor, nontumorous colorectal tissue and blood demonstrate that the immunosuppressive/exhausted T cells were recruited/induced by the tumor microenvironment, even at the early stage of tumor development. To improve the response of immunotherapy targeting immune checkpoints, it is important to reverse the immunosuppressive tumor microenvironment.…”
Section: Discussionmentioning
confidence: 92%
“…In contrast, a previous study found that patients with breast cancer exhibited most PD-1+ T cells within the CD8+ compartment, whereas the mean expression of PD-1 was higher in CD4+ than in CD8+ T cells. 25 The mean expression level of PD-1 and PD-1 T-cell frequency strongly correlated with CD4+ and CD8+ subsets, indicating that these cells originated from T-cell expansion (Fig. 3e).…”
Section: T-cell Phenotypes Associated With Immunosuppression At the Tmentioning
confidence: 84%
“…RNA‐sequencing determines the mRNA levels of huge number of genes in the bulk of tumor, while mass cytometry is a powerful single‐cell proteomic analysis technique. By compiling and then comparing the levels of expression of many proteins across multiple cell types, single‐cell mass cytometry is a robust readout to clarify the differentiation of expression patterns in multiple immune cell types …”
The majority of patients with microsatellite stable (MSS) colorectal cancer (CRC) do not benefit from the immunotherapies directed at rescuing T‐cell functions. Therefore, complete understanding of T‐cell phenotypes and functional status in the CRC microenvironment is desirable. Here, we applied single‐cell mass cytometry to mold the T‐cell phenotype in 18 patients with MSS CRC for better understanding of CRC as a systemic disease and to search for tumor‐driven T‐cell profile changes. We show interpatient and intrapatient phenotypic diversity of T‐cell subsets. We revealed increased immunosuppressive/exhausted T‐cell phenotypes at tumor lesions. CD8+ CD28− immunosenescent T cells with impaired proliferation capacity dominate the T‐cell compartment. As per the transcriptome and quantitative real time‐PCR analysis, the accumulation of immunosuppressive cells is driven by the tumor microenvironment. T‐cell profiles are similar between patients at early and late stages, indicating that the immunosuppressive microenvironment is formulated early during CRC development. Mapping of T‐cell infiltration and understanding of the mechanisms underlying their regulation may provide valuable information to boost the immune response in patients with MSS CRC.
“…The outcome showed that programmed cell death‐ligand 1 positive (PD‐L1 + ) tumor‐associated macrophages and exhausted T cells were abundant in high‐grade ER + and ER − tumors. This comprehensive single‐cell atlas of breast tumor indicated the significance of ecosystem‐based patient classification for precision medicine approaches …”
The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed.
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