Chemotherapy-induced increase in CD47 expression in epithelial ovarian cancer

Introduction

Ovarian cancer is a most lethal gynecological malignancy and has a 5-year survival rate of approximately 40%. There were an estimated 324,398 new cases of ovarian cancer and 206,839 ovarian cancer-related deaths globally in 2022 (1). Moreover, ovarian cancer is fifth leading oncological cause of death among women of all ages in the United States (2). Epithelial ovarian cancer (EOC) is the most common pathological type of ovarian cancer. At the time of diagnosis, the majority of patients present with advanced-stage disease, exhibiting insidious onset, delayed detection, chemoresistance, and a propensity for recurrence. The treatment of ovarian cancer has long been based on surgical intervention and platinum-based chemotherapy (3). The application of poly (ADP-ribose) polymerase (PARP) inhibitors and bevacizumab has led to an improvement in the clinical outcomes of some patients (4). However, given the high lethality of this type of malignant tumor, there is a continued need to explore more effective treatment options. Anticancer immunotherapy, exemplified by immune checkpoint inhibitors such as programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1), has advanced considerably in recent years (5). However, it has yet to yield promising outcomes in ovarian cancer, an immune cold tumor (6). Upregulation of coinhibitory molecules, notably PD-L1, suggests an immunosuppressive response to chemotherapy (7). To achieve optimal efficacy, new targets, combined therapies and a more precise timing of administration are essential, and the innate immune checkpoint CD47 seems to be an attractive candidate target.

CD47 is an immunoglobulin superfamily glycoprotein that is widely expressed on the cell surface and is overexpressed on the cell surface of a variety of tumors, including ovarian cancer (8). In this context, it interacts with platelet-reactive protein-1 [also known as signal regulatory protein alpha (SIRP-α)] and other factors to regulate cellular functions, including cell migration, axonal extension, cytokine production, and T-cell activation. CD47 has been identified as a highly expressed protein in a range of solid tumors, including ovarian cancer (9), and its presence has been linked to a poor prognosis for patients with these cancers (8). CD47 inhibits the phagocytosis of tumor cells by macrophages by binding to SIRP-alpha, which is predominantly expressed on the surface of macrophages. This process facilitates the evasion of tumor cells from immunosurveillance (9). Given its pivotal function as a negative checkpoint in innate and adapted immunity, the CD47-SIRPα axis has been identified as a promising new target for tumor immunotherapy (10,11), with multiple clinical trials currently underway (12,13). Although combination therapy targeting the CD47–SIRPα axis with other antibody drugs or therapies has demonstrated promising antitumor efficacy, the widespread expression of CD47 in erythrocytes, myeloid cells, and other hematopoietic cells presents a significant challenge to CD47-targeted drug therapy, as it can lead to anemia (14). Research into the immune microenvironment in ovarian cancer has indicated that chemotherapy may lead to immunosuppression by increasing the expression of factors such as PD-L1 (7). It is therefore of particular importance to select the appropriate patients and to determine the optimal timing of treatment. However, considerable advancements need to be made before immunotherapy involving the targeting of the CD47-SIRPα axis can be applied in the clinic.

In this retrospective study, we examined patients with EOC who had not undergone targeted therapy to avoid the influence of other targeted therapy. With the hypotheses that chemotherapy could change the expression of CD47 in ovarian cancer, we investigated the expression of CD47 and its relationship with the clinicopathological characteristics and prognosis of these patients, with particular attention given to the effect of chemotherapy on the expression of CD47 in EOC. In vitro cytological studies were conducted with the objective of initially investigating the underlying cause of this chemotherapy-induced alteration in CD47 expression at the cytological level. We present this article in accordance with the REMARK and MDAR reporting checklists (available at https://gs.amegroups.com/article/view/10.21037/gs-24-400/rc).

Methods

Patients

The cases were selected from 78 consenting patients who were diagnosed with ovarian, fallopian tube, or primary peritoneal cancer between 2010 and 2012 at the Department of Gynecology and Obstetrics at Peking Union Medical College Hospital (PUMCH). All patients received the standard initial surgery with the platinum-based neoadjuvant chemotherapy (NACT) and an interval debulking surgery (IDS). The patients were identified retrospectively via a database search from the PUMCH archives, and a confirmed histological diagnosis of EOC was obtained. Additionally, the patients had available sequential samples taken before and after NACT. The primary exclusion criteria were the presence of poor-quality or inadequate material (for example, insufficient remaining tumor tissue volume or tissue preservation), incomplete clinical information or a lack of follow-up data, and a previous history of immune system disease or malignancies. The aforementioned database provided access to comprehensive clinical information for each patient. The medical records of the patients were also retrospectively reviewed in accordance with the requirements of an approved institutional protocol. The review encompassed both outpatient and inpatient treatment, including surgical and chemotherapeutic procedures.

All studies concerning human specimens were approved by the Ethics Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (No. ZS-1771) and were conducted in accordance with the Declaration of Helsinki (as revised in 2013). Informed consent was taken from all individual participants.

Specimen characteristics

The pre-NACT samples were obtained from a biopsy conducted at the time of diagnosis, during laparoscopy or following primary surgery. Post-NACT samples were obtained during the IDS. The Department of Pathology provided formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemical analysis. All specimens were reviewed by two independent pathologists at our institution, with histopathologic subtype classification conducted in accordance with World Health Organization (WHO) guidelines.

Study design

The end of follow-up for all patients was May 2018, and the median follow-up time was 44 months. The study endpoint and outcomes included overall survival (OS), time to progression, serum CA125 levels both pre- and post-NACT, and surgical information. Progression was defined as the presence of objective evidence of recurrence. The recurrence of the disease was defined as an increase in the CA125 level or the emergence of new lesions as observed through imaging techniques. Chemotherapy-sensitive recurrence was defined as a recurrence occurring at least 6 months after postoperative chemotherapy. Chemotherapy-resistant recurrence was defined as recurrence occurring less than 6 months after postoperative chemotherapy. Finally, chemotherapy-refractory disease was defined as a disease that progressed or was stable during the initial chemotherapy. OS was defined as the time elapsed between diagnosis and death. The follow-up period was defined as the interval between the initial diagnosis and the final contact, which could be either patient’s death or the last follow-up. In this study, we considered the following candidate variables for inclusion in the prognostic model: age, International Federation of Gynecology and Obstetrics (FIGO) stage, histological type, pathological grade, NACT regimen, number of NACT cycles, pretreatment CA125 level, percentage of CA125 decrease after treatment, postoperative residual tumor status, and CD47 expression level. Due to the retrospective design of this study, the sample size was determined based on patients EOC who were treated at our institution between 2010 and 2012 and had complete follow-up data. This sample size reflected the range of data available to us and allowed us to perform meaningful statistical analyses although it might have been affected by selection bias. We acknowledge the limitations of the sample size and have considered the impact this may have on the interpretation of the study results in the Discussion.

Assay methods

Immunohistochemical (IHC) staining for CD47

An IHC analysis was conducted using FFPE tissues from 78 patients. Tissue sections with a thickness of 5 µm were deparaffinized and pretreated in EDTA at a pH of 9.0 (cat. no. G1203; Guge Biotechnology Co., Ltd., Wuhan, China) with a microwave oven. Following a 20-minute cooling period, the slides were washed with phosphate-buffered saline (PBS; pH 9.0) and incubated for 10 minutes with 3% hydrogen peroxide to quench endogenous peroxidase activity. A 30-minute incubation with a bovine serum albumin (BSA) protein block (cat. no. A8020; Solarbio, Beijing, China) was conducted to achieve effective blocking. Anti-CD47 polyclonal antibody (cat. no. AF4670; R&D Systems, Minneapolis, MN, USA) was added at a dilution of 1:150 to each section and incubated overnight at 4 ℃ (15).

IHC scoring

For assessment of CD47 expression, the stained tissue slides were evaluated at 40× magnification for a minimum of five distinct sites. The slides were scored based on membranous and cytoplasmic staining of CD47 antibody. A scale of 0–3 was employed as follows: 0, ≤25% staining; 1, >25–50% positivity; 2, >50–75% positivity and cells showing membranous and/or cytoplasmic expression; and 3, intense membranous and/or cytoplasmic expression of at least 75% of the tissue section (16). The scoring was conducted in a fully double-blind manner, and a consensus score was reached between two reviewers in the event of a discrepancy. Additionally, as CD47 exhibits a predominantly membranous staining pattern, with no nuclear staining observed, only ovarian cancer cell staining was considered, and the red blood cells served as the inner positive control (17).

In vitro cell experiments

In our experiments, both technical and biological replications were performed to ensure the reliability and generalizability of the results. For technical replicates, at least three technical replicates were performed for each experimental condition to ensure consistency of operation and precision of results. For biological replicates, we used two different types of ovarian cancer cell lines to assess the variability of CD47 expression in different pathological types of ovarian cancer. With this design, we were able to distinguish between technical and biological variability in the experimental results to more accurately assess the changes of CD47 in ovarian cancer after chemotherapy.

Cell lines and culture

Human ovarian surface epithelial cell line (HOSE) and the human ovarian cancer cell SKOV3 and ES2 cells were purchased from the Cell Center of the Institute of Basic Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, and cultured according to the provider’s guidelines.

Drug treatment of ovarian cancer cells with cisplatin

SKOV3 and ES2 cell lines in logarithmic growth phase were obtained and inoculated in six-well plates, and when the cells grew to a confluence of about 20–30%, the medium was aspirated, the cells were gently washed with PBS twice, different concentrations of cisplatin-complete medium solutions were added, and the cells were placed into an incubator at 37 ℃ with 5% CO₂ for incubation. A control group was treated with 0 µM of cisplatin, while a second group was treated with 4 µM of cisplatin for 48 hours.

Determination of cisplatin half maximal inhibitory concentration in ovarian cancer cells

SKOV3 and ES2 cells in the logarithmic growth phase were washed twice with PBS, trypsin digested, and centrifuged at 800 rpm, after which the supernatant was discarded. The resulting single-cell suspension was prepared via addition of complete medium and inoculated into 96-well plates with 3,500 cells/well. Twenty-four hours later, once the cells had attached to the wall, the supernatant was aspirated and discarded. The cells were then added to a 200 µL/well gradient of cisplatin-complete medium and incubated at 37 ℃ with 5% CO₂ for 48 hours. Following a 48-hour incubation period, the cell proliferation was determined with cell counting kit 8 (CCK-8) assay according to the manufacturer’s protocol (Engreen Biosystem Ltd., Beijing, China).

RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR)

The RNA was extracted from the cell lines using TRIzol Reagent (Solarbio). Total RNA was extracted using an RNA isolation kit (Dakewe, Shenzhen, China). RT-qPCR was performed using the Takara RT-PCR kit (Takara Bio, Kusatsu, Japan). The RT-qPCR experiments were conducted in triplicate. The primers specific for CD47 and GAPDH were purchased from Tsingke Biotech (Beijing, China). The sequences of the primers employed were as follows: CD47 primer forward, 5'-TCCGGTGGTATGGATGAGAAA-3'; CD47 primer reverse, 5'-ACCAAGGCCAGTAGCATTCTT-3'; GAPDH primer forward, 5'-ACAACTTTGGTATCGTGGAAGG-3'; and GAPDH primer reverse, 5'-GCCATCACGCCACAGTTTC-3'. Target messenger RNA (mRNA) expression was normalized to GAPDH mRNA expression (18).

Flow cytometry

The cells were stained with an FITC-conjugated anti-human CD47 antibody (cat. no. CC2C6; BioLegend, San Diego, CA, USA) in accordance with the manufacturer’s instructions. The staining procedure was conducted at 4 ℃ for 30 minutes in PBS in the absence of light. Following a period of washing, the samples were subjected to immediate analysis by flow cytometry with an Accuri C6 Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) (19).

Statistical analysis

Categorical variables were compared using a chi-square test, while continuous variables were compared using t-tests, ANOVA tests and Wilcoxon exact tests, with the Wilcoxon signed-rank tests being used for paired ranked samples. Fisher’s exact tests were employed as appropriate. The Kaplan-Meier curves were employed to generate survival curves. Cox regression analyses were used for multifactorial analyses. The statistical analyses were conducted using SPSS 23 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA) software. A P value of less than 0.05 was considered statistically significant.

In this study, CD47 expression levels were assessed via IHC scoring based on the degree of staining in the cell membrane and/or cytoplasm, with scores ranging from 0 to 3. The cutoff points for CD47 expression were based on values used in previous literature (16) and validated in our dataset to differentiate between the high and low expression groups. Specifically, an IHC score of 0–1 was defined as low CD47 expression and an IHC score of 2–3 as high CD47 expression. In multivariate analyses, CD47 expression was included in the model as a categorical variable to assess its independent association with prognostic factors. We also performed sensitivity analyses to assess the effect of different cutoff points on the model results.

Results

Overview of patient flow

A total of 78 patients were included in this study, all of whom received NACT and IDS at our institution between 2010 and 2012. Initially, in the enrolment phase, 100 patients were identified who met the inclusion criteria. Of these, 22 patients were excluded due to lack of follow-up data, leaving a total of 78 patients in the study. In the pretreatment phase, the remaining 78 patients underwent surgery or tumor tissue biopsy prior to NACT, and ovarian cancer samples and baseline CD47 expression data were obtained. In the NACT phase, after completion of NACT, 78 patients received IDS. In the postoperative phase, all patients provided postoperative tumor tissue samples following IDS. Valid tumor tissue and CD47 expression data were not available for three of these samples due to treatment response, and these samples were excluded from the paired analysis. During a median follow-up of 44 months, we recorded the OS and progression-free survival (PFS) in all patients, and four patients without advanced ovarian cancer were excluded from the survival analysis.

Patient population

A total of 78 pairs of tissues from patients with ovarian, primary peritoneal, or fallopian tube cancer who had undergone NACT and IDS were subjected to IHC analysis. The characteristics of the patients included in this study are presented in Table 1. The majority (74/78, 94.9%) of patients presented with advanced-stage disease. Four patients were classified as stage I–II, while 74 were stage III–IV. The majority (57/78; 73.1%) of patients exhibited serous histology, followed by Mullerian epithelial carcinoma, clear cell, and mixed endometrioid–clear cell carcinoma (Table 1).

Expression of CD47 in ovarian cancer

The expression of CD47 was evaluated through IHC analysis (Figure 1). CD47 expression was observed in 63 of the 78 (80.8%) pre-NACT ovarian cancer specimens, and in 67 of the 75 (89.3%) specimens post-NACT. Subsequently, the patients were classified according to the degree of pre-NACT CD47 expression as CD47 high (IHC score 0–1) or CD47 low (IHC score 2–3). The median age of the two groups was 52 and 57 years, respectively (Table 2), and no significant difference was observed in tumor grade, histological type, platinum sensitivity, FIGO stage, CA125 pre-NACT, CA125 pre-IDS, percentage of reduction of CA125 and residual disease post-IDS between the two groups (P>0.05), (Table 2).

Figure 1 Expression of CD47 in epithelial ovarian cancer tissues (100×) according to IHC staining. Specimens were stained with anti-CD47 polyclonal antibody. CD47 exhibits a predominantly membranous staining pattern, with no nuclear staining observed. A scale of 0–3 was employed: 0: ≤25% staining, 1: >25–50% positivity, 2: >50–75% positivity, cells showed membranous and/or cytoplasmic expression, 3: intense membranous and/or cytoplasmic expression of at least 75% of the tissue section. (A) CD47 IHC staining score of 0; (B) CD47 IHC staining score of 1; (C) CD47 IHC staining score of 2; (D) CD47 IHC staining score of 3. IHC, immunohistochemical.

Change in CD47 expression after NACT

Three of the cancer specimens were excluded from the study due to the absence of cancer cells following NACT. In the evaluation of all available paired tumors, using a cutoff of 25% to define CD47 positivity, the proportion of cases with CD47-positive cells was 81.3% (61/75) at diagnosis and significantly higher at 89.3% (67/75) post-NACT (Table 3, Figure 2). The expression of CD47 was found to increase significantly following NACT (P=0.006) (Table 4).

Figure 2 Change of CD47 expression pre- and post-NACT. NACT, neoadjuvant chemotherapy.

Survival analysis

Four cases of stage I and II ovarian cancer were excluded from the analysis. The median OS was 38 months in the group with low pre-NACT CD47 expression vs. 37 months in the group with high pre-NACT CD47 expression, which did not reach statistical significance (P=0.76). The median PFS was 9 months in both the groups with low pre-NACT CD47 expression and high pre-NACT CD47 expression (P=0.59) (Figure 3). The expression level of pre-NACT CD47 was not associated with the OS or PFS of in our cohort of patients. Cox regression analysis was performed for multifactorial analysis with age being the stratification factor, and the cutoff value was 60 years old. The results suggested that the pathological grade 3 (P=0.02), platinum-refractory (P<0.001) and platinum-resistant (P=0.001) present independent adverse prognostic factors for ovarian cancer, while the pre-NACT CD47 expression level was not an independent prognostic factor for ovarian cancer (Table 5). We conducted the omnibus test for model coefficients.

Figure 3 Kaplan-Meier curves of CD47 expression for OS and PFS. The figure shows the Kaplan-Meier curves of OS and PFS for CD47 high and low expression in the cohort of 74 patients with EOC who received neoadjuvant chemotherapy, and four cases of stage I and II ovarian cancer were excluded from the analysis. EOC, epithelial ovarian cancer; OS, overall survival; PFS, progression-free survival; NACT, neoadjuvant chemotherapy.

Cisplatin half maximal inhibitory concentration in ovarian cancer cell lines

Following the addition of a cisplatin concentration gradient to a 96-well plate, the proliferation rate of each cell line was assessed at 48 hours. The absorbance values were used to plot the curves with GraphPad Prism 10, and the half maximal inhibitory concentration (IC₅₀) values for cisplatin in the ovarian cancer cell lines were determined as shown in Table 6. Accordingly, the experimental concentration was set at 4 µM.

The expression of CD47 mRNA in HOSE cells and ovarian cancer cells

The differential expression of CD47 mRNA in HOSE cells and ovarian cancer cells was assessed through RT-qPCR. Cells in the logarithmic growth phase were subjected to this analysis. The mRNA expression levels of CD47 in ES2 and SKOV3 cells were found to be significantly higher than those in HOSE cells (P<0.001, Figure 4).

Figure 4 The expression of CD47 mRNA in ovarian epithelial cells (HOSE) and ovarian cancer cells (SKOV3 and ES2) according to RT-qPCR analysis of CD47 mRNA levels. The levels of CD47 mRNA expression in ES2 and SKOV3 cells were found to be significantly higher than those in HOSE cells (P<0.001). ***, P<0.001. RT-qPCR, reverse transcription quantitative polymerase chain reaction; mRNA, messenger RNA; HOSE, human ovarian surface epithelial cell.

Increased CD47 mRNA expression in SKOV3 cells and ES2 cells after cisplatin treatment

The SKOV3 and ES2 cells treated with cisplatin at a concentration of 4 µM for 48 hours exhibited varying degrees of CD47 mRNA changes (Figure 5). The expression of CD47 mRNA was found to be significantly elevated in both SKOV3 and ES2 cells that had been treated with 4 µM of cisplatin for a period of 48 hours. Cisplatin induced a significant increase in the transcription of the CD47 gene in both SKOV3 cells (P<0.001) and ES2 cells (P<0.001).

Figure 5 The CD47 mRNA expression in SKOV3 and ES2 cells both with and without cisplatin treatment. A control group was treated with 0 µM of cisplatin, while a second group was treated with 4 µM of cisplatin for 48 hours to assess the expression of CD47 mRNA. (A) The expression of CD47 mRNA in SKOV3 treated with 0 and 4 µM cisplatin for 48 hours. Following 48 hours of treatment with 4 µM of cisplatin, a notable elevation in CD47 mRNA was observed (P<0.001). (B) The expression of CD47 mRNA in ES2 treated with 0 and 4 µM of cisplatin for 48 hours. Following a 48-hour treatment with 4 µM of cisplatin, a notable elevation in CD47 mRNA was evident (P<0.001). ***, P<0.001. mRNA, messenger RNA; DDP, cisplatin.

Flow cytometry analysis of cell surface CD47 protein expression in SKOV3 and ES2 cells with and without cisplatin treatment

SKOV3 and ES2 cells were cultured under standard conditions for 48 hours. The 0-µM group consisted of SKOV3 or ES2 cells cultured under normal conditions for 48 hours. The 4-µM group consisted of SKOV3 or ES2 cells cultured in a medium containing 4 µM of cisplatin for the same period. After a 48-hour cisplatin treatment period, the mean fluorescence intensity of the surface CD47 protein in both SKOV3 and ES2 cells demonstrated a notable increase (P<0.001) (Figure 6).

Figure 6 Expression of cell surface CD47 protein in SKOV3 and ES2 cells following cisplatin treatment. (A) Following a 48-hour cisplatin treatment period, the mean fluorescence intensity of the surface CD47 protein in SKOV3 cells demonstrated a statistically significant increase (P<0.001). (B) Following a 48-hour cisplatin treatment period, the mean fluorescence intensity of the surface CD47 protein in ES2 cells demonstrated a notable increase (P<0.001).

Discussion

Ovarian cancer remains the most lethal gynecological malignancy, with a low 5-year survival rate that has prompted a constant search for newer and better treatments. The recent development of PARP inhibitors may significantly improve the outcomes of ovarian cancer (20,21). In recent years, immunotherapy has yielded promising outcomes in the field of anticancer therapy, particularly in the context of immune checkpoint inhibitors, such as those targeting PD-1/PD-L1 (22). Despite the fact that ovarian cancer is in principle immunoreactive, the efficacy of immune checkpoint inhibitors in ovarian cancer has thus far failed to meet expectations, with the mean response rate to PD1 or PD-L1 blockade therapy ranging between 10% and 15% (23).

CD47, another prominent immune checkpoint, is an immunoglobulin-like transmembrane receptor with a multitude of physiological functions, including cell migration, activation of T cells and dendritic cells, and axon development (24). Binding of CD47 to thrombospondin-1 also influences angiogenesis and perfusion. The ligation of CD47 to SIRPα transmits inhibitory signals that prevent phagocytosis by macrophages, thereby enabling tumor cells to evade immune detection and proliferation. This unique inhibitory property of CD47 represents the first intrinsic immune checkpoint to be identified, and it is currently a highly active area of research, following the lead of work on PD-L1. It has been demonstrated that CD47 is markedly expressed in a number of human malignancies, including EOC, and is associated with a range of adverse clinicopathological factors (25). Chemo-resistance in tumor stem cells is a major challenge in tumor therapy. CD47 expression was found to be higher in CSCs (cancer stem cells) of HCC, lung cancer, and pancreatic ductal adenocarcinoma in clinical investigations. CD47 interacts with signal regulatory protein (SIRP-α) on TAMs to inhibit phagocytosis of CSCs (26). However, Chang et al. established a mice ovarian cancer model with the cancer stem-like cells, and found them susceptible to phagocytosis by macrophages and consequent CD8⁺ T cell immunity due to the low CD47 protein expression. SCA-1 + ID8 cells were able to grow in syngeneic mice but were soon rejected. Restoring CD47 expression delayed this immune-mediated rejection (27). A considerable number of studies have been conducted on immune checkpoint inhibitors targeting the CD47-SIRPα axis, with a significant proportion of these undergoing clinical investigation (28-32). A study conducted by Yang et al. revealed that PPAB001 markedly enhanced the phagocytosis of tumor cells by macrophages via the concurrent inhibition of the CD47/SIRPα and CD24/Siglec-10 signaling pathways. The increase in the M1:M2 ratio in tumor-infiltrating macrophages in PPAB001-treated mice indicates that dual blockade may facilitate the transition of macrophages from M2 to M1 (33). Chen et al. constructed HER2 and CD47 chimeric antigen receptor (CAR) macrophages for the treatment of ovarian cancer and observed that CAR macrophages enhanced the activation of CD8⁺ T cells, affected the phenotype of tumor-associated macrophages (TAMs), and resulted in tumor regression (34). However, due to its extensive expression on the surface of erythrocytes, the significant adverse effects it causes restrict its clinical utilization. Selecting the appropriate patients and determining the optimal timing for clinical studies is particularly critical.

Mesnage et al. observed elevated expression of invasive T lymphocytes and PD-L1 in paired pre- and post-NACT samples of EOC, suggesting that chemotherapy may increase the likelihood of immune escape related to ovarian cancer survival (35). Furthermore, assessment of these immune markers after NACT may assist in selecting appropriate patients for immunotherapy clinical trials. Ongoing trials exploring neoadjuvant immune checkpoint inhibitors and chemotherapy offer promise for advanced ovarian cancer treatment (7). In our study, CD47 expression in ovarian cancer tissues was significantly elevated following NACT (P=0.006). This may be associated with the immune escape and drug resistance mechanisms of tumors after chemotherapy; moreover, these insights may inform the theoretical foundation for the identification of patients who may benefit from immunotherapy targeting the CD47–SIRPα axis, the selection of appropriate treatment modalities, and the timing of treatment.

Our study revealed that CD47 is highly expressed in ovarian cancer, which is consistent with the findings of Yu et al. (36) and Luo et al (37). However, the effect of CD47 on the prognosis of ovarian cancer remains controversial. In the majority of related studies, CD47 has been identified as an independent risk factor for a poor prognosis in ovarian cancer. Yu et al. detected the expression of CD47 in ovarian cancer tissues with IHC, thereby demonstrating that patients with low CD47 expression exhibited superior prognoses compared to those with high CD47 expression (36). Luo et al. employed IHC and next-generation sequencing to characterize the expression of CD47 in various malignant biological and genetic characteristics of EOC. Their findings suggest that CD47 may play a pivotal and multifaceted role in the tumor microenvironment of EOC. Moreover, they found that patients with high CD47 expression exhibited a poorer prognosis than did those with low expression, and CA125, CD47, and BRCA mutation were identified as independent prognostic factors for EOC (37). Meanwhile, Brightwell et al. reported that there was no difference in survival between patients with ovarian cancer with different CD47 expression, either in their exploratory analysis of The Cancer Genome Atlas (TCGA) data or in a subsequent retrospective study. It is worth noting that theirs is the most comprehensive retrospective study to date to examine the prognostic impact of CD47 in patients with ovarian cancer (38). Our study did not find there to be a correlation between CD47 expression and the prognosis of patients with ovarian cancer. This discrepancy may be related to the fact that our study cases were mainly patients who received NACT for advanced ovarian cancer and were therefore excluded from the group of patients who typically have a better prognosis without residual tumors postoperatively. Consequently, we did not find a correlation between prognosis and positive results in this group of samples. The aim of this study was to observe the effect of chemotherapy on the expression of CD47 in ovarian cancer, and the main method of the study was self-controlled. However, as a nonrandomized controlled retrospective study, there might have been selection bias in the enrolled patients due to the limitation in obtaining specimens, resulting in the common indicators of poor prognosis, such as stage and postoperative residual tumor, not showing significant differences in the Cox multifactorial analysis.

The results of our in vitro cellular studies demonstrated that CD47 was expressed at varying levels in ovarian epithelial cell lines and ovarian cancer cell lines, with notable distinctions observed in CD47 mRNA and cell surface CD47 protein expression. Following treatment with the chemotherapeutic drug cisplatin, the expression of CD47 mRNA and cell surface CD47 protein in ovarian cancer cells SKOV3 and ES2 was markedly elevated, with a notable disparity. These findings suggest that cisplatin chemotherapy may lead to the elevation of CD47 transcription in ovarian cancer cells, which contributes to the elevation of CD47 protein expression in cell membranes. This may be due to an increase in CD47 mRNA transcription and a change in the distribution position of CD47 protein, which could promote the immune escape of cancer cells.

In vitro cellular experiments provided a partial corroboration of the phenomenon of elevated CD47 expression after chemotherapy observed in the clinical tissue specimens. This may be attributed to two factors. First, chemotherapy only facilitates the migration of CD47 to the cell membrane. Second, the complexity of the immune factors in the immunological microenvironment of in vivo interactions in the immune microenvironment may be a contributing factor.

CD47 as an immunotherapeutic target has been extensively studied in solid tumors and haematological tumors (32), including ovarian cancer, and a large number of clinical trials are unfolding for monoclonal antibodies targeting the CD47-SIRPα pathway, but the vast majority of them are directed at advanced patients who have repeatedly failed treatment. Based on our results that CD47 is elevated after chemotherapy, immunotherapy targeting CD47 in combination with chemotherapy (synchronous or sequential therapy) may be a promising new direction compared with prior single-agent CD47-targeted immunotherapy. Of course, the clinical application of this approach needs to be supported by more and more in-depth studies.

This study was subject to certain limitations, including limited sample and the inability to ascertain the genetic features. Unfortunately, as a retrospective study, due to the limitation of sample selection and sample acquisition, genetic testing for ovarian cancer patients was not yet as regular as it is now, none of our patients were tested for genes such as BRCA, HRD status, TP53, etc., which is a pity. And at the same time, the specimens and the results were not affected by other targeted therapies. It would have been possible to use these as stratification factors in order to further analyze the relationship between CD47 and the prognosis and clinical characteristics of ovarian cancer. More studies, especially those including in vivo experiments, are needed to analyze the mechanism by which cisplatin chemotherapy leads to altered CD47 expression in ovarian cancer and how cisplatin treatment affects the immune environment. This could provide a research foundation for combination targeted therapy and help to determine an optimal timing for immunotherapy.

Conclusions

The findings of our study indicate that CD47 is highly expressed in ovarian cancer and that NACT can have a significant effect on the expression of the immune evasion marker CD47, which may contribute to tumor immune evasion. It is recommended that the impact of chemotherapy on the tumor immune microenvironment be assessed from a variety of research perspectives. Furthermore, additional studies are required to elucidate the interaction between chemotherapy and the immune phenotype of immune cell subsets in order to determine the most appropriate criteria for selecting patients eligible for immune checkpoint inhibitors and the optimal timing for their administration.

Acknowledgments

We appreciate the pathologists from the Department of Pathology for the IHC slides reading and the gynecologists of PUMCH for their diligent clinical work in the cases we reported in this article.

Funding: None.

Footnote

Reporting Checklist: The authors have completed the REMARK and MDAR reporting checklists. Available at https://gs.amegroups.com/article/view/10.21037/gs-24-400/rc

Data Sharing Statement: Available at https://gs.amegroups.com/article/view/10.21037/gs-24-400/dss

Peer Review File: Available at https://gs.amegroups.com/article/view/10.21037/gs-24-400/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-24-400/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All studies concerning human specimens were approved by the Ethics Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (No. ZS-1771) and were conducted in accordance with the Declaration of Helsinki (as revised in 2013). Informed consent was taken from all individual participant.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. [Crossref] [PubMed]
2. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin 2024;74:12-49. [Crossref] [PubMed]
3. Kehoe S, Hook J, Nankivell M, et al. Primary chemotherapy versus primary surgery for newly diagnosed advanced ovarian cancer (CHORUS): an open-label, randomised, controlled, non-inferiority trial. Lancet 2015;386:249-57. [Crossref] [PubMed]
4. Hirschl N, Leveque W, Granitto J, et al. PARP Inhibitors: Strategic Use and Optimal Management in Ovarian Cancer. Cancers (Basel) 2024;16:932. [Crossref] [PubMed]
5. Yang Y. Cancer immunotherapy: harnessing the immune system to battle cancer. J Clin Invest 2015;125:3335-7. [Crossref] [PubMed]
6. Le Saux O, Ray-Coquard I, Labidi-Galy SI. Challenges for immunotherapy for the treatment of platinum resistant ovarian cancer. Semin Cancer Biol 2021;77:127-43. [Crossref] [PubMed]
7. Spagnol G, Ghisoni E, Morotti M, et al. The Impact of Neoadjuvant Chemotherapy on Ovarian Cancer Tumor Microenvironment: A Systematic Review of the Literature. Int J Mol Sci 2024;25:7070. [Crossref] [PubMed]
8. Majeti R, Chao MP, Alizadeh AA, et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 2009;138:286-99. [Crossref] [PubMed]
9. Weiskopf K. Cancer immunotherapy targeting the CD47/SIRPα axis. Eur J Cancer 2017;76:100-9. [Crossref] [PubMed]
10. Feng M, Jiang W, Kim BYS, et al. Phagocytosis checkpoints as new targets for cancer immunotherapy. Nat Rev Cancer 2019;19:568-86. [Crossref] [PubMed]
11. Logtenberg MEW, Scheeren FA, Schumacher TN. The CD47-SIRPα Immune Checkpoint. Immunity 2020;52:742-52. [Crossref] [PubMed]
12. Voets E, Paradé M, Lutje Hulsik D, et al. Functional characterization of the selective pan-allele anti-SIRPα antibody ADU-1805 that blocks the SIRPα-CD47 innate immune checkpoint. J Immunother Cancer 2019;7:340. [Crossref] [PubMed]
13. Bouwstra R, van Meerten T, Bremer E. CD47-SIRPα blocking-based immunotherapy: Current and prospective therapeutic strategies. Clin Transl Med 2022;12:e943. [Crossref] [PubMed]
14. Isenberg JS, Montero E. Tolerating CD47. Clin Transl Med 2024;14:e1584. [Crossref] [PubMed]
15. Xu Y, Li J, Tong B, et al. Positive tumour CD47 expression is an independent prognostic factor for recurrence in resected non-small cell lung cancer. ESMO Open 2020;5:e000823. [Crossref] [PubMed]
16. Sakakura K, Takahashi H, Kaira K, et al. Relationship between tumor-associated macrophage subsets and CD47 expression in squamous cell carcinoma of the head and neck in the tumor microenvironment. Lab Invest 2016;96:994-1003. [Crossref] [PubMed]
17. Yang Z, Xu J, Li R, et al. PD-L1 and CD47 co-expression in pulmonary sarcomatoid carcinoma: a predictor of poor prognosis and potential targets of future combined immunotherapy. J Cancer Res Clin Oncol 2019;145:3055-65. [Crossref] [PubMed]
18. Xu CR, Li JR, Jiang SW, et al. CD47 Blockade Accelerates Blood Clearance and Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage. Front Immunol 2022;13:823999. [Crossref] [PubMed]
19. Pascual-Pasto G, McIntyre B, Shraim R, et al. GPC2 antibody-drug conjugate reprograms the neuroblastoma immune milieu to enhance macrophage-driven therapies. J Immunother Cancer 2022;10:e004704. [Crossref] [PubMed]
20. Colombo N, Ledermann JAESMO Guidelines Committee. Electronic address: clinicalguidelines@esmo. Updated treatment recommendations for newly diagnosed epithelial ovarian carcinoma from the ESMO Clinical Practice Guidelines. Ann Oncol 2021;32:1300-3. [Crossref] [PubMed]
21. DiSilvestro P, Banerjee S, Colombo N, et al. Overall Survival With Maintenance Olaparib at a 7-Year Follow-Up in Patients With Newly Diagnosed Advanced Ovarian Cancer and a BRCA Mutation: The SOLO1/GOG 3004 Trial. J Clin Oncol 2023;41:609-17. [Crossref] [PubMed]
22. Zhang C, Yang Q. Predictive Values of Programmed Cell Death-Ligand 1 Expression for Prognosis, Clinicopathological Factors, and Response to Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Inhibitors in Patients With Gynecological Cancers: A Meta-Analysis. Front Oncol 2020;10:572203. [Crossref] [PubMed]
23. Hamanishi J, Mandai M, Ikeda T, et al. Safety and Antitumor Activity of Anti-PD-1 Antibody, Nivolumab, in Patients With Platinum-Resistant Ovarian Cancer. J Clin Oncol 2015;33:4015-22. [Crossref] [PubMed]
24. Tan M, Zhu L, Zhuang H, et al. Lewis Y antigen modified CD47 is an independent risk factor for poor prognosis and promotes early ovarian cancer metastasis. Am J Cancer Res 2015;5:2777-87.
25. Wang H, Tan M, Zhang S, et al. Expression and significance of CD44, CD47 and c-met in ovarian clear cell carcinoma. Int J Mol Sci 2015;16:3391-404. [Crossref] [PubMed]
26. Bayik D, Lathia JD. Cancer stem cell-immune cell crosstalk in tumour progression. Nat Rev Cancer 2021;21:526-36. [Crossref] [PubMed]
27. Chang CL, Wu CC, Hsu YT, et al. Immune vulnerability of ovarian cancer stem-like cells due to low CD47 expression is protected by surrounding bulk tumor cells. Oncoimmunology 2020;9:1803530. [Crossref] [PubMed]
28. Luo X, Shen Y, Huang W, et al. Blocking CD47-SIRPα Signal Axis as Promising Immunotherapy in Ovarian Cancer. Cancer Control 2023;30:10732748231159706. [Crossref] [PubMed]
29. Nishiga Y, Drainas AP, Baron M, et al. Radiotherapy in combination with CD47 blockade elicits a macrophage-mediated abscopal effect. Nat Cancer 2022;3:1351-66. [Crossref] [PubMed]
30. Zhang X, Fan J, Wang S, et al. Targeting CD47 and Autophagy Elicited Enhanced Antitumor Effects in Non-Small Cell Lung Cancer. Cancer Immunol Res 2017;5:363-75. [Crossref] [PubMed]
31. Gholamin S, Mitra SS, Feroze AH, et al. Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors. Sci Transl Med 2017;9:eaaf2968. [Crossref] [PubMed]
32. Liu Y, Weng L, Wang Y, et al. Deciphering the role of CD47 in cancer immunotherapy. J Adv Res 2024;63:129-58. [Crossref] [PubMed]
33. Yang Y, Wu H, Yang Y, et al. Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy. Mol Ther Oncolytics 2023;31:100747. [Crossref] [PubMed]
34. Chen Y, Zhu X, Liu H, et al. The application of HER2 and CD47 CAR-macrophage in ovarian cancer. J Transl Med 2023;21:654. [Crossref] [PubMed]
35. Mesnage SJL, Auguste A, Genestie C, et al. Neoadjuvant chemotherapy (NACT) increases immune infiltration and programmed death-ligand 1 (PD-L1) expression in epithelial ovarian cancer (EOC). Ann Oncol 2017;28:651-7. [Crossref] [PubMed]
36. Yu L, Ding Y, Wan T, et al. Significance of CD47 and Its Association With Tumor Immune Microenvironment Heterogeneity in Ovarian Cancer. Front Immunol 2021;12:768115. [Crossref] [PubMed]
37. Luo X, Mo J, Zhang M, et al. CD47-a novel prognostic predicator in epithelial ovarian cancer and correlations with clinicopathological and gene mutation features. World J Surg Oncol 2024;22:44. [Crossref] [PubMed]
38. Brightwell RM, Grzankowski KS, Lele S, et al. The CD47 "don't eat me signal" is highly expressed in human ovarian cancer. Gynecol Oncol 2016;143:393-7. [Crossref] [PubMed]