Breast cancer stem cells generate immune-suppressive T regulatory cells by secreting TGFβ to evade immune-elimination

Abstract

Cancer stem cells (CSCs), being the primary contributors in tumor initiation, metastasis, and relapse, ought to have seminal roles in evasion of immune surveillance. Tumor-promoting CD4⁺CD25⁺FOXP3⁺ T-regulatory cells (Tregs) have been described to abolish host defense mechanisms by impeding the activities of other immune cells including effector T cells. However, whether CSCs can convert effector T cells to immune-suppressive Treg subset, and if yes, the mechanism underlying CSC-induced Treg generation, are limitedly studied. In this regard, we observed a positive correlation between breast CSC and Treg signature markers in both in-silico and immunohistochemical analyses. Mirroring the conditions during tumor initiation, low number of CSCs could successfully generate CD4⁺CD25⁺FOXP3⁺ Treg cells from infiltrating CD4⁺ T lymphocytes in a contact-independent manner. Suppressing the proliferation potential as well as IFNγ production capacity of effector T cells, these Treg cells might be inhibiting antitumor immunity, thereby hindering immune-elimination of CSCs during tumor initiation. Furthermore, unlike non-stem cancer cells (NSCCs), CSCs escaped doxorubicin-induced apoptosis, thus constituting major surviving population after three rounds of chemotherapy. These drug-survived CSCs were also able to generate CD4⁺CD25⁺FOXP3⁺ Treg cells. Our search for the underlying mechanism further unveiled the role of CSC-shed immune-suppressive cytokine TGFβ, which was further increased by chemotherapy, in generating tumor Treg cells. In conclusion, during initiation as well as after chemotherapy, when NSCCs are not present in the tumor microenvironment, CSCs, albeit present in low numbers, generate immunosuppressive CD4⁺CD25⁺FOXP3⁺ Treg cells in a contact-independent manner by shedding high levels of immune-suppressive Treg-polarizing cytokine TGFβ, thus escaping immune-elimination and initiating the tumor or causing tumor relapse.

Keywords: Anti-tumor immunity; Cancer stem cells; Metastasis; Microenvironment; Relapse; Stemness markers; TGFβ; Treg cells; Tumor initiation.

Fig. 1

Tumor-initiating CSCs and immunosuppressive Treg cells have positive correlation in breast cancer. A Plots showing correlation between NANOG and FOXP3, OCT4 and FOXP3, from GSE25066 breast cancer patient dataset (left). Plots demonstrating positive correlation between cancer stemness marker ALDH1A1 and suppressive Treg signature FOXP3 in brain cancer glioma (dataset used: Tumor Brain Lower Grade Glioma (2022-v2)-tcga-532-tpm-gencode36) (middle) and prostate cancer (dataset used: Tumor Prostate Adenocarcinoma-TCGA-497-rsem-tcgars) (right). Datasets were obtained from ‘R2: Genomics Analysis and Visualization Platform’ and ‘TCGA’ database. “r” is correlation co-efficient. B Kaplan-Meier (KM) plot depicting low overall survival (OS) probability of breast tumor patients harboring high expression of CSC marker NANOG and Treg signature gene FOXP3. C Representative Immunohistochemistry (IHC) images showing elevated expression of stemness markers OCT4, SOX2, NANOG, and Treg signature gene FOXP3 in low-grade vs. high-grade breast tumor tissues (n = 4 in each group). Scale bar = 50 µM and magnification 40X. D Bar diagram showing number of OCT4, SOX2, NANOG, and FOXP3 positive cells in low-grade vs. high-grade breast tumor tissues as determined by IHC-staining. E Bar graphs showing incidence of higher cancer stemness markers OCT4, SOX2, NANOG, and ALDH1A1 along with suppressive Treg marker FOXP3 in TNBC tissues than ER⁺-luminal breast tumor tissues. Bar graphs were plotted using RNA profiling data of TNBC dataset (GSE30682) and ER⁺-luminal breast tumor dataset (GSE5460) available in ‘R2: Genomics Analysis and Visualization Platform’. F Flow-cytometry data showing percent CD44⁺CD24⁻ CSC population in ER⁺-luminal MCF-7, triple-negative MDA-MB-231, and MDA-MB-468 breast cancer cells. G Plot showing a positive correlation between ALDH1A1 and FOXP3 in TNBC dataset (GSE76714) available in ‘R2: Genomics Analysis and Visualization Platform’. H Bar graph demonstrating occurrence of higher Treg cell percentage in TNBC patients than non-TNBC patients. Data were represented as the mean ± SD of minimum 3 independent experiments performed in triplicate. Student’s t-test (unpaired) (D, E, H) and one-way ANOVA (F) was used to assess the data where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. BC: breast cancer, TNBC: triple-negative breast cancer

Fig. 2

Low number of CSCs are sufficient to generate immunosuppressive Treg cells. A Inverted microscopic image showing the appearance of monolayer MDA-MB-468 cells and cell-derived spheroids viewed under 10X magnification. B Schematic representation of immunosuppressive Treg cell generation using CSC-CM taking single-cultured CSCs and T cells in 1:5 ratio. C FACS plots (left panel) and representative graph (right panel) showing immunosuppressive Treg cell percentage in (i) only α-CD3 + α-CD28, (ii) α-CD3 + α-CD28 + TGFβ + IL2, and (iii) α-CD3 + α-CD28 + CSC-CM. D Flow-cytometric histoplots (left panel) and representative bar graph (right panel) showing T-cell proliferation in presence of CSC-induced Treg cells. E FACS plots (left panel) and bar graph (right panel) furnishing percentage of IFNγ secreting CD4⁺ T cells after co-culture with CSC-induced Treg cells. Data were represented as the mean ± SD of minimum 3 independent experiments performed in triplicate. Student’s t-test (unpaired) was used to assess the data where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. CSCs: cancer stem cells; CM: conditioned medium

Fig. 3

Doxorubicin-spared CSCs induce immunosuppressive Treg cells mimicking tumor-initiation following tumor relapse. A Bar graph showing percentage of apoptotic NSCCs and CSCs populations gated from MDA-MB-468 cells after doxorubicin treatment, as determined using flow-cytometry. B Schematic representation of three cycles of doxorubicin treatment regimen to MDA-MB-468 cell-derived spheroids. C Bar diagrams depicting percentage of apoptotic NSCCs and CSCs after 1st cycle (left), 2nd cycle (middle), and 3rd cycle (right) of chemotherapy. Percent apoptosis was flow-cytometrically determined using Annexin-V binding assay. D Representative bar diagram showing percentage of CD44⁺/CD24⁻ CSCs in control vs. post- 3 cycle doxorubicin-treated MDA-MB-468-derived spheroids (left). Bar plot showing total number of cells counted using hemocytometer from control vs. post-3 cycledoxorubicin-treated spheroids. E Schematic representation of Treg cell generation using MDA-MB-468 spheroid-derived CSC-CM and CSC-CM after 3 cycles of doxorubicin treatment. F Flow-cytometric plots showing percentage of Treg cells (left) and bar graph (right panel) showing percentage of suppressive Treg cells generated from activated T cells in presence of doxorubicin-treated or untreated MDA-MB-468 spheroid-derived CSC-CM. Data were represented as the mean ± SD of minimum 3 independent experiments performed in triplicate. Student’s t-test (unpaired) was used to assess the data where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. CSCs: cancer stem cells; Sph: spheroids; CM: conditioned medium

Fig. 4

CSC-secreted TGFβ induces immunosuppressive Treg cell generation. A Plot showing positive correlation between CSC markers ALDH1A1 and TGFβ1, from GSE69031 breast cancer patient dataset (left) and representative plot demonstrating correlation between TGFβ1 and suppressive Treg marker FOXP3 using GSE5460 breast cancer dataset (right panel). Datasets were obtained from ‘R2: Genomics Analysis and Visualization Platform’ database. “r” is correlation co-efficient. B Plots showing a positive correlation of ALDH1A1 with TGFβ (left panel) and FOXP3 with TGFβ (right panel) in TNBC dataset (GSE142102) available in ‘R2: Genomics Analysis and Visualization Platform’. C Representative bar plots showing level of secreted TGFβ in the CM of MDA-MB-468-derived CSCs and NSCCs, as determined by ELISA. D FACS plots (left panel) and bar diagram (right panel) depicting percent immune-suppressive Treg cells generated from CD4⁺ T cells following activation by (i) only α-CD3 + α-CD28, (ii) α-CD3 + α-CD28 + TGFβ + IL2, and (iii) α-CD3 + α-CD28 + CSC-CM. E FACS plots (left panel) and bar diagram (right panel) depicting percent Treg cell generation from activated T cells cultured in presence of CSC-CM pre-incubated with or without anti-TGFβ antibody. F Bar plot demonstrating expression of TGFβ per-CSC in control vs. 3-cycles of doxorubicin-treated CSCs derived from MDA-MB-468 spheroids. G Representative bar plot showing the amount of TGFβ present in the CM of control vs. 3-cycles of doxorubicin-treated CSCs derived from MDA-MB-468 spheroids determined by ELISA. Data were represented as the mean ± SD of minimum 3 independent experiments performed in triplicate. Student’s t-test (unpaired) was used to assess the data where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. CSC: cancer stem cell; MFI: mean fluorescence intensity; CM: conditioned medium