Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, because it does not express hormone receptors as well as HER2, can not use targeted therapy, for a long time TNBC patients can only undergo surgery and radiotherapy and chemotherapy.
The advent of immune checkpoint inhibitor therapy has given new hope to patients with TNBC, but clinical trials have found that PD-1 inhibitors combined with chemotherapy benefit only a small percentage of patients with TNBC [1,2]. Therefore, it is important to explore the mechanism of TNBC resistance to immune checkpoint inhibitor therapy, which will provide a reference for improving the effectiveness of immunotherapy.
Recently, a research team led by Professor Judith Agudo of Dana-Farber Cancer Center published an important study in the journal Cell[3], and they found that there is a group of resting tumor cells (QCCs) in TNBC that resist T cell killing. By activating HIF1a, these QCCs, together with immunosuppressive fibroblasts and dysfunctional DC cells, form a microenvironment that resists T cell infiltration and killing. Thus surviving immunotherapy and causing tumor recurrence.
The results of this study unveil the mystery of breast cancer immune escape, highlight the important role of QCCs in breast cancer immune escape, and suggest that targeting the elimination of QCCs may be an effective way to crack the problem of TNBC immune resistance.
Screenshot of the first page of the paper
Previous studies have found that some genes play a role in tumor cell immunotherapy [4,5], but these studies do not explain how tumor cells create an immunosuppressive microenvironment; in addition, sequencing can find some of the characteristics of immune checkpoint inhibitors for the treatment of drug-resistant tumors, but there are a large number of heterogeneous cell populations in tumor tissue, which greatly increases the difficulty of finding tumor resistance mechanisms. For example, cancer cells that lose tumor antigens are obviously different from cancer cells that are positive for tumor antigens in the process of escaping the immune system.
In this study, Professor Agudo's team hopes to explore how cancer cells that express tumor antigens escape the immune system. Therefore, they inoculated the mice with ATNBC cell line 4T07 expressing GFP or mCherry and transfused GFP-specifically recognized PD-1-/-Jedi T cells for mice (knockout PD-1 was done to mimic PD-1 inhibitor therapy). Since the TCR of Jedi T cells can only specifically recognize GFP and not mCherry. Therefore, this experimental model can be used to explore how tumor cells (GFP+ tumor cells) that continuously express tumor-specific antigens can escape attack by tumor antigen-specific T cells (Jedi T cells).
The research team analyzed the ratio of GFP+ to mCherry+ tumor cells in the tumor on the fifth day after transfusion of Jedi T cells, and found that Jedi T cells killed most of the GFP+ tumor cells, but still some GFP+ tumor cells escaped the T cell's killing. Immunofluorescence staining found that these GFP+ tumor cells that escaped killing formed a cluster of cells, with T cell infiltration levels twice reduced inside the cluster. This suggests that surviving clusters of GFP+ tumor cells are able to block infiltration of T cells, which may be one of the reasons for their resistance to immunotherapy.
GFP+ tumor cells that survive after Jedi T cells attack aggregate into clusters
Next, Professor Agudo's team analyzed transcriptome differences between surviving GFP+ tumor cells and control mCherry+ tumor cells. They found that genes associated with cell cycles in GFP+ tumor cells were significantly downregulated compared to mCherry+ tumor cells. EdU (thymine nucleoside analogue 5-acetylene-20-deoxyuridine) incorporation experiments showed that these GFP+ tumor cells that survived Jedi T cell attack were in a state of cell cycle arrest (called resting tumor cells, QCCs).
So are these QCCs more resistant to the killing of T cells? The research team transferred tdTomato-p27K into tumor cells, which allowed QCCs to be tagged with tdTomato. Transfusion of PD-1-/- Jedi T cells to tumor-bearing mice found that most of the surviving GFP+ tumor cells were tdTomato+ compared with the control non-immunogenic miRFP670+ tumor cells, indicating that QCCs have stronger resistance to T cell killing.
GFP+ tumor cells that survive Jedi T cell attacks are in a cell cycle resting state and are more resistant to T cell killing
The above results show that in mouse models, QCCs are the main force of immunotherapy resistance, so does this phenomenon also exist in human patients? By analyzing histopathological samples of breast cancer patients and transcriptome sequencing, the research team found that most of the tumor cells in contact with T cells in tumor tissue were Proliferating cells of Ki67+, and the QCCs of p27+ rarely came into contact with T cells; while gene collection enrichment analysis showed that the tumor cells of immunotherapy responders were more enriched in DNA replication-related pathways than those who did not respond to treatment, indicating that they were in a non-quiescent state. This correlation suggests that QCCs may be associated with poor immunotherapy in breast cancer patients.
Patients with breast cancer have less infiltration of T cells around resting tumor cells in tumor tissue, and patients who respond to immunotherapy have tumor cells in a state of cell cycle activity
To explore the mechanism of drug resistance in QCCs, the research team sequenced RNA from QCCs as well as non-quiescent tumor cells. Data analysis showed that QCCs upregulated genes related to hypoxia and glucose metabolism. Immunofluorescence staining showed that p27K+QCCs were co-localized with hypoxic probes, while T cells rarely infiltrated into hypoxic regions; GFP+ tumor cells that survived After Jedi T cell therapy were also located in hypoxic regions. These data suggest that QCCs in breast cancer are mainly located in hypoxic microenvironments with less infiltration of T cells.
The region where the QCCs are located is in a significantly hypoxic state and there is less T cell infiltration
Next, to analyze the mechanisms by which QCCs inhibit T cell function at the single-cell level, Professor Agudo's team isolated infiltrating cells inside and outside the cluster of QCCs and sequenced them in a single-cell transcriptome.
Through data analysis, it was found that a large number of immunosuppressive fibroblasts are present inside the QCCs cell cluster, which explains why the QCCs region infiltrates a small number of T cells.
The analysis of infiltration T cells inside and outside the QCCs cell cluster found that the CD8+ T cells infiltrated inside the hypoxic QCCs cell clusters were more depleted and less killing ability.
There are a large number of immunosuppressive fibroblasts inside the QCCs cluster, the number of infiltrated T cells is significantly lower than outside the QCCs cluster, and the infiltration of T cells in the QCCs cluster is more depleted
Which group of cells promotes dysfunction of T cells? By scoring the hypoxia characteristics of each cell subpopulation infiltrated inside QCCs, the research team found that DC cells expressed rich hypoxia-related genes and downregulated genes that promoted T cell activation, such as MHCI. and MHCII., IL-12, CD80/86, indicating that the ability of these DC cells to promote T cell activation was greatly weakened, which may be an important reason for the dysfunction of T cells infiltrated inside QCCs cell clusters.
However, Professor Agudo's team found through hypoxia induction experiments that hypoxia does not directly weaken the ability of DC cells to activate T cells, so the research team turned its attention to QCCs themselves. It was explored whether HIF1a-expressing QCCs shaped the immunosuppressive microenvironment, impairing the ability of DC cells to activate T cells and thereby inhibiting T cell immune responses. To do this, they constructed breast cancer cell lines expressing activated HIF1a (HIF1aSTBL).
Tumor-bearing experiments showed that the number of infiltration T cells in the tumor expressing HIF1aSTBL was smaller and the depletion was deeper than that of WT tumors, and the expression of MHCII. on intratumorAL DC cells was significantly downregulated, which was basically consistent with the observed inside the QCCs cell cluster.
Further, the research team inoculated WT or HIF1aSTBL-expressing GFP+ tumor cells with mCherry+ tumor cells into mice subcutaneously. Compared with the mCherry+: GFP+ tumor group, mice inoculated with mCherry+: GFP+ HIF1aSTBL tumor cells had fewer intratumorous T cells, and T cells were more difficult to approach GFP+ tumor cells, and the number of surviving GFP+ tumor cells increased several times, which showed that HIF1a could promote immune escape of tumor cells.
Tumor cells overexpress HIF1aSTBL can inhibit the infiltration of T cells and escape attack by T cells
Based on the above data, it can be seen that QCCs create an immunosuppressive microenvironment through high expression of HIF1a, escaping the pursuit of tumor antigen-specific CD8+ T cells. So can knocking out HIF1a in tumor cells promote the killing of tumors by T cells? The experimental results show that after knocking out HIF1a in tumor cells, the number of infiltrating T cells in the tumor increases significantly, the degree of depletion decreases, and the tumor volume decreases significantly. This suggests that targeting intratumoral HIF1a can promote an anti-tumor immune response and inhibit tumor growth.
Knockout of HIF1a in tumor cells significantly enhances the anti-tumor immune response
Overall, the study sheds light on the mechanism by which cancer cells that are tumor antigen-positive escape: they aggregate into clumps and summon fibroblasts to form a barrier for them; they enter a resting state together and express HIF1a highly, making the sentinel of the immune system, the DC cells, dysfunctional, and thus suppressing the T-cell immune response. These efforts allowed them to escape the T cells and make a comeback after immunotherapy.
At the same time, the results of this study also suggest that eradication of QCCs is the key to improving the effectiveness of TNBC immunotherapy and preventing tumor recurrence.
Bibliography:
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Responsible editor 丨Ying Yuyan