Senescent Cell Escape and Drug Resistance in Breast Cancer Chemotherapy

During the experiment, 250 nM doxorubicin was used to treat MCF7 cells for 2 hours, then the drug was withdrawn, and the medium was replaced with drug-free medium for continuous culture. We found that the aging cells gradually apoptotic, which lasted until about the 10th day and reached the 15th day. There were re-proliferated senescent escape clonal clusters, and as time went on, the monoclonal clusters gradually increased (Fig. 2A).

Next, we picked out the senescent escape clones in good growth state to re-culture, and named the cells MCF7-T (M7-T for short), and then performed SA-β-Gal staining, and found that the M7-T Staining was negative (Figure 2B). Cell cycle experiments showed that compared with the senescent cell M7-SE, the escaped cell M7-T escaped from G2/M phase arrest, the ratio of G2/M phase decreased, the S phase increased, and the cycle distribution was similar to that of the parental normal MCF7, indicating that senescence Cells escaped senescence-induced cycle arrest and reproliferated after doxorubicin withdrawal (Fig. 2C). In addition, we also compared the IC50 of normal MCF7, senescent M7-SE, and senescent escape M7-T cells through CCK-8 experiments, and the results showed that M7-SE was more drug-resistant (Fig. 2D).

We know that p21 and p16 are highly expressed in senescent cells.^16 At the protein level, compared with senescent M7-SE, the expression of p21 and p16 of M7-T was decreased, and the expression of Lamin B1 was up-regulated, indicating that M7-T escaped from cellular senescence (Fig. 2E). Therefore, we speculate that doxorubicin-induced senescent cells are related to drug resistance after breast cancer chemotherapy, while escaped senescent cells are related to drug-resistant recurrence.

Screening for Targeted Markers of senescent

MCF7 cells were treated with 250 nM doxorubicin for 2 h, and then replaced with drug-free medium. The untreated MCF7 (Con) cells, the senescent MCF7 (M7-SE) cells on the 5th day after drug withdrawal, and the aged MCF7 (M7-SE) cells were extracted respectively. For the mRNA of the escaped cells (M7-T), Real-time PCR was used to detect the differences in the transcription levels of the three aging-related markers, and they were displayed in the form of a heat map (Fig. 3 A). The results showed that DCR2, p21, p16 and other indicators were significantly expressed in the aging group (M7-SE). Next, we compared the protein levels of the above indicators. The Western Blot results of the three groups of cells showed that the protein levels of DCR2, p21 and p16 were significantly different, which can be used as candidate markers of senescent cells (Fig. 3 B).

In addition, since p21 and p16 are expressed in the nucleus^15, 17, they are not suitable as target markers for subsequent ultrasound molecular probes, while DCR2 protein is mainly expressed on the cell membrane^18. At the same time, by immunofluorescence experiments, we further verified the cellular localization and expression of DCR2 in doxorubicin-induced cells M7-SE and normal MCF7 (Fig. 3C). The nuclei were stained with DAPI, and DCR2 was marked with FITC. The results showed that DCR2 was significantly highly expressed on the cell membrane of senescent cell M7-SE.

Doxorubicin induces senescence of breast cancer cells and causes M2 polarization of macrophages in the immune microenvironment

Regarding the reason for the changes in the immune microenvironment in the process of doxorubicin resistance in breast cancer, we speculate that it may be closely related to senescent cells. Drug-induced senescent tumor cells can secrete a series of soluble factors, including pro-inflammatory cytokines, chemokines, growth factors, proteases and biologically active exosomes, namely the cellular senescence-associated secretory phenotype (SASP).^19 SASP is not a single phenotype but is highly complex, dynamic, with composition that changes dramatically over time and depends on senescence inducers and cell type.^20 SASP can generate a pro-inflammatory environment and recruit immune cells, activate the tumor immunosuppressive network, such as myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) and M2 macrophages, promote tumorigenic immunosuppressive microenvironment, and then Accelerates tumor initiation, progression, and metastasis, leading to inefficiency in immunotherapy.^21 Macrophages are divided into M1 type and M2 type, in which M1 macrophages can phagocytose tumor cells, on the contrary, M2 macrophages have the ability to promote tumor growth and invasion.^22 In order to explore the relationship between tumor cell senescence induced by chemotherapy resistance in breast cancer and the polarization of macrophages in the immune microenvironment, we used PMA to transform THP1 cells into M0 macrophages after stimulation, and then combined with doxorubicin to induce senescence in breast cancer cells. Cells M7-SE were co-incubated for 48 hours, and the co-incubation pattern is shown in (Fig. 4A). The proportion of M0 macrophages polarized into M2 macrophages after co-incubation was detected by flow cytometry. We found that compared with the blank control THP1 group and the untreated tumor cell co-incubation THP1+MCF7 group, the morphology of THP1 co-incubated with senescent cells M7-SE was significantly changed, and the proportion of strip-like and bifurcated morphologies increased. That is, the polarization ratio increases (Fig. 4B). Next, we conducted a flow cytometry experiment, using APC-Cy7-labeled CD163 to represent M2-type TAMs. The results showed that THP1 and CD163 indicators were significantly up-regulated and the proportion of M2-type macrophages was significantly increased when incubated with senescent cells M7-SE (Fig. 4C). The results showed that while doxorubicin induced the senescence of breast cancer cells, the polarization ratio of M2 TAMs in the immune microenvironment changed significantly. In order to further verify the relationship between breast cancer chemotherapy resistance, aging and changes in the immune microenvironment in vivo, we then conducted an in vivo verification on MMTV-PyMT mice with spontaneous tumor formation, and the blank control group was injected with normal saline intraperitoneally, In addition, two groups of mice were set up. Both groups were treated with doxorubicin intraperitoneally at a concentration of 100 mg/ml, and each mouse was injected with 4 mg/kg according to body weight. One week later, they were executed as the senescence resistance group (Senescence) after chemotherapy; the other group stopped the drug for 4 weeks, and were executed when the tumor volume increased again, which was simulated as the drug-resistant recurrence group (Recurrence) after chemotherapy. During the administration period, the body weight and tumor volume of the mice were recorded, and the hearts, liver, spleen, lungs, kidneys, and tumors of the mice were collected after sacrifice for immunohistochemical staining (Fig.4D-F). The results showed that the tumor volume increased after withdrawal of the drug, obvious lung metastases appeared in the relapse group after aging, the aging indicators DCR2 and γh2AX increased, and the M2 macrophage marker CD163 increased.