Angiotensin-converting Enzyme Inhibitors Have Adverse Effects in Anti-angiogenesis Therapy for Hepatocellular Carcinoma

At present, Anti-angiogenic drugs (AADs) are widely used in the systemic treatment of hepatocellular carcinoma (HCC) or other types of cancer, and have achieved good anti-cancer effect, whereas treatment-related proteinuria can affect the routine use of AADs, which in turn abates the overall efficacy. Currently, most clinicians prescribe angiotensin-converting enzyme inhibitors (ACEIs) to alleviate proteinuria according to diabetic nephropathy guidelines or expert recommendations. However, the efficacy of ACEIs in reducing AAD-related proteinuria and its effect on the anticancer effect of AADs is unknown. Our clinical data showed that some HCC patients experienced tumor progression by ACEI administration for the treatment of proteinuria caused by AADs. Here, we confirmed that in different tumor-bearing mouse models, ACEIs did not delay the appearance of proteinuria or alleviate proteinuria caused by AADs but compromised the anticancer efficacy of AADs. This effect is unrelated to a change in the VEGF signaling pathway. Our data showed that the combination of ACEIs and AADs flared the production of kidney-derived erythropoietin (EPO). In turn, EPO compromises the anti-angiogenic effects of AADs and decreases antitumor activity. In conclusion, for the treatment of proteinuria caused by AADs, ACEIs have no efficacy while also promoting AADs resistance. This finding is of great significance to guide clinical standardized management of side effects of anti-angiogenic therapy for cancer patients.

tumor progression by ACEIs administration for the treatment of hypertension or proteinuria caused by AADs. We have con rmed that in different tumor-bearing mouse models, ACEIs did not delay the appearance of proteinuria or alleviate proteinuria caused by AADs but compromised the anticancer e cacy of AADs. This effect is unrelated to a change in the VEGF signaling pathway. Our data showed that the combination of ACEIs and AADs ared the production of kidney-derived erythropoietin (EPO). In turn, EPO compromises the anti-angiogenic effects of AADs and decreases antitumor activity.
Conclusions: For the treatment of proteinuria caused by AADs, ACEIs have no e cacy but promote drug resistance. Kidney-derived EPO is mainly responsible for ACEIs induced anti-angiogenesis resistance.

Background
Approximately half of patients with HCC have reached an advanced stage, and in these patients, systemic drug therapy is the most important option for prolonging survival. HCC is a typical blood vesselrich tumor, and anti-angiogenesis-related therapies have become the standard treatment for patients with advanced HCC [1][2][3]. To date, the US Food and Drug Administration has approved 4 oral tyrosine kinase inhibitors (TKIs) and 1 anti-angiogenic antibody for the treatment of advanced HCC [4]. Meanwhile, the combined application of immune checkpoint inhibitors and TKIs is the current clinical research trend, and the results are promising [5,6]. Whether used alone or in combination with other drugs, AADs will become the main choice for systemic treatment of HCC in the near future.
AADs improve the overall survival of patients with HCC, but their side effects may require the administered dose to be reduced, thereby limiting the clinical bene t [7]. The three side effects of the most concern are hand-foot syndrome, hypertension and proteinuria [8]. Except for proteinuria, hand-foot syndrome and hypertension can be well controlled, and good management strategies for these side effects have been developed according to previous clinical experience. It has been reported that AADrelated proteinuria occurs in 30% to 70% of patients [9][10][11]. Many patients undergo dose reduction or temporary stop use of AADs, which in turn leads to compromised therapeutic effects. Therefore, good management of AAD-related proteinuria plays a vital role in ensuring the e cacy of anti-angiogenic therapy and the stable development of new therapies targeting this pathway [8]. However, regarding tumor anti-angiogenic therapy, nephrotoxicity associated with pharmacological inhibition of VEGF signaling in the kidney and its downstream pathways remains a concern. Although the side effects of AAD-related proteinuria have been well described in numerous experimental and clinical studies, to date, no strategy has been established to reduce proteinuria [12][13][14].
Currently, most clinicians prescribe ACEIs to treat proteinuria caused by AADs according to diabetic nephropathy guidelines, expert recommendations or drug instructions to reduce the occurrence or degree of proteinuria. However, there is no de nitive evidence showing that ACEIs reduce the proteinuria caused by AADs. Moreover, it remains controversial whether ACEIs promote cancer. Some studies show that the use of ACEIs is closely related to tumor progression and a low survival rate. However, most studies have relatively small sample sizes and short follow-up times, and some are designed to assess overall cancer risk but not to speci cally assess the risk of a certain type of cancer [15][16][17]. Hicks et al. conducted a large cohort study including one million people and con rmed the use of ACEIs associated with increased overall lung cancer risk [18]. Wysocki et al. found that captopril promotes the growth of immunogenic tumors and promotes tumor recurrence after surgery in a mouse model of renal cell carcinoma [19]. Emile et al. found that ACEIs reduce the e cacy of bevacizumab in the treatment of ovarian cancer [20]. Based on these results, we question whether ACEIs are suitable for the treatment of AAD-related proteinuria.
Nakamura et al. con rmed that hypoxia in the kidneys induced by AADs would increase circulating EPO levels [21]. Some studies have shown that EPO protects tumor blood vessels from damage by AADs and thereby reduces its antitumor effect, blocking EPO function can inhibit tumor angiogenesis and signi cantly increase the antitumor activity of AADs [21,22]. Pradeep et al. discovered another EPO receptor, erythropoietin HCC type B receptor 4 (EphB4). It can trigger downstream signaling and promote EPO-induced tumor growth and progression [23]. The above studies reveal that EPO is the key to weakening the anticancer effect of AADs. The renin-angiotensin system (RAS) is an endocrine system of peripheral blood and has been established as a key regulator of blood pressure and steady-state volume.
Some studies have proven that angiotensin II has a close relationship with EPO [24,25].
Here, we demonstrate using two different tumor-bearing mouse models that ACEIs do not interfere with the effect of AADs on the VEGF signaling pathway or with the anti-angiogenic effects of the kidney, which indicates that ACEIs have no e cacy for delaying or reducing proteinuria caused by AADs. We further show that ACEIs compromise the anticancer e cacy of AADs without affecting the VEGF signaling pathway. Finally, we provide evidence that ACEIs promote AAD-induced renal EPO production, which may be a cause of AADs resistance. Mice were killed after the experimental observations. Necropsy was performed, the tumor tissues, kidneys, liver and lungs were removed, and the quality of the kidneys and tumor were measured. A fraction of the tissues and organs was immediately xed with 4% (wt/vol) paraformaldehyde (PFA) overnight, followed by washing with PBS and embedding with para n until further use. Blood samples were collected from all animals, and serum samples were kept at −80°C until further use.

Urine analyses
Metabolic cages were used to collect 24-hour urine from mice one time per week. The gross albuminuria of each group was detected using Coomassie staining of 10 μl of urine run on an SDS-PAGE gel.
Quantitative analysis was measured using an ELISA kit (Bethyl, USA), and urine creatinine was measured using a colorimetric assay kit (Exocell, USA) following the manufacturer's methodology.

Histological evaluation
Para n-embedded tissues were sectioned at a thickness of 3 μm and stained with hematoxylin and eosin (H&E) or Periodic acid Schiff (PAS) and Masson's trichrome staining kits (Sigma, USA), according to standard techniques. Tissues were imaged with a universal upright uorescence microscope and imaging system (OLYMPUS BX61, USA). The glomerular damage and liver and lung metastatic tumor nodules on each slice were evaluated and counted by an experienced pathologist, and the proportion of damaged glomeruli was calculated. The mean glomerular volume area was calculated based on the average volume of 30 glomeruli in each group, and the equation GV = (β/κ) ×GA 3/2 (1), where β = 1.38, the shape coe cient of spheres (the idealized shape of glomeruli), κ = 1.1, the size distribution coe cient, and GA is the glomerular area [26].

Fresh-frozen immuno uorescence staining and imaging
Frozen sections of tissues were prepared by the Department of Pathology of our hospital. Fresh-frozen tumor and kidney sections were xed in methanol (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., China) for 20 minutes at -20°C. Then, the sections were incubated with blocking solution containing 1% bovine serum albumin (BSA) and 5% FBS in PBS at room temperature for 3 h. Tissues were incubated overnight with primary antibodies diluted in blocking solution. We used rabbit polyclonal antibodies against CD31 (Abcam, ab28364, USA) as primary antibodies. After 3 washes for 5 min each with blocking solution, samples were incubated with secondary antibodies diluted in PBS for 1 h. The secondary antibody was goat anti-rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081, USA) from Abcam. Samples were washed 3 times with PBS and subsequently stained with DAPI to reveal nuclei, which were diluted in PBS at a concentration of 1 μg/ml. Sections were mounted in Dako Fluorescence Mounting Medium, covered with a cover glass, and stored at 4°C. Images were captured with a universal inverted uorescence microscope (Leica DMI6000B, USA), and ImageJ software was used for image analysis and quanti cation.
EPO ELISA Serum levels of mouse EPO were quanti ed by using an ELISA method according to the instructions (Abcam, ab119522, USA).

Statistical analysis
GraphPad Prism 8.0 (GraphPad Prism Software, La Jolla, CA, USA) was used for statistical analyses. All values are expressed as the mean ± SD. For analyses of two groups, we performed T-tests if data had normal distributions; for analyses of more than two groups, we performed ANOVA with Tukey's test for multiple comparisons or Sidak's test for comparisons against a single control group. The Kaplan-Meier method was used to analyze the survival of mice. All statistical tests were performed as two-tailed analyses, and P < 0.05 was considered statistically signi cant.

Results
Captopril does not delay or alleviate proteinuria caused by apatinib and may counteract the anticancer e cacy of apatinib in HCC patients and tumor-bearing mouse models Apatinib, a speci c small molecule tyrosine kinase inhibitor (TKI), can highly selectively inhibit the tyrosine kinase activity of vascular endothelial growth factor receptor 2 (VEGFR-2). In China, apatinib has been recommended as second-line treatment of advanced gastric cancer and HCC [28][29][30]. From 2016 to the present, we used single-agent apatinib to treat 208 patients with advanced HCC. Apatinib exhibited a convincing anticancer effect and acceptable toxicity [31,32]. We found that Captopril did not reduce the proteinuria caused by apatinib, and several patients even experienced deterioration. Patient 1 with severe advanced HCC received apatinib for 26 months and was in clinical, radiological and biological partial response when she developed severe hypertension and proteinuria. She was then administered captopril and experienced progression within the next 18 months. Alpha-fetoprotein (AFP) showed an upward trend throughout the entire captopril administration time and an occasional decrease because of the doubled dose of apatinib. The dose had to be reduced frequently because of intolerance. Captopril did not prevent the production of severe proteinuria caused by apatinib during the entire use process. Eventually, after the patient stopped using apatinib, the proteinuria level gradually decreased (Figure 1a). Patient 2 was also in radiological and biological partial response after 3 months of apatinib treatment. In the fourth month, she began taking captopril due to hypertension and proteinuria, and she then experienced progressive disease. After discontinuing captopril, the response to apatinib resumed (Supplemental Figure 1, a and c).
The proteinuria was not alleviated by the use of captopril. Patient 3 had a partial response (PR) after 2 months of apatinib treatment. Subsequently, the patient was administered captopril at the same time due to proteinuria, and the disease progressed (Supplemental Figure 1, b).
Next, we subcutaneously injected MHCC-97H cells labeled with luciferase into the lateral forearm of BALB/c (nu/nu) male mice. Once tumors were established, the mice were given apatinib (200 mg/kg) daily, and the bioluminescence intensity of the mice was detected weekly. One week of apatinib administration decreased the luminescence signal by more than 60%, but the signal recovered after one week of supplementation with captopril (30 mg/kg/day) and decreased again after captopril was stopped for a week. The same trend was found in the next two dosing cycles (Figure 1b). These data suggest that ACEIs do not delay or reduce the proteinuria caused by AADs but rather may counteract the anti-angiogenic effect of AADs.
Determining the dose of drug used in the tumor-bearing mouse model and establishing a stable proteinuria model related to AADs Because captopril was reported to promote tumor growth in an immunogenic model and decrease the survival of immunogenic mice in a dose-dependent manner [33], we established two mouse subcutaneous tumor models, the immunogenic C57BL/6 mouse model with Hep1-6 cells and the immunode cient BALB/c nude mouse model with MHCC-97H cells to verify this effect. To generalize the role of ACEIs, we chose three representative drugs: captopril containing thiol, enalapril without thiol and fosinopril containing phosphorus [34]. After tumors were established (Figure 2, a and e), the mice received different doses of ACEIs [vehicle, high-dose captopril (60 mg/kg/day), low-dose captopril (30 mg/kg/day), highdose enalapril (60 mg/kg/day), low-dose enalapril (30 mg/kg/day), high-dose fosinopril (10 mg/kg/day) and low-dose fosinopril (5 mg/kg/day)], which were based on the low and high doses used in humans according to a body surface area dose conversion method, as reported in previous literature [19,35,36]. The high dose was designed to be twice the low dose because the recommended dose of ACEIs for the treatment of proteinuria is twice that for the treatment of hypertension according to diabetic nephropathy guidelines [37,38]. We observed that ACEIs decreased the survival of tumor-bearing immunogenic mice in a dose-dependent manner (Supplemental Figure 2a). However, in immunode cient tumor-bearing mice, we found that only high-dose ACEIs reduced survival time, whereas low-dose ACEIs did not affect survival time (Supplemental Figure 2b). Regardless of immunogenic or immunode cient tumor-bearing mice, high and low doses of ACEIs did not affect tumor growth (Supplemental Figure 2, c and d). These results prompted us to choose low-dose ACEIs in subsequent animal experiments.
The doses of apatinib in the treatment of tumor-bearing mice were 50 mg/kg/day, 100 mg/kg/day and 200 mg/kg/day according to the previous literature [39][40][41]. A comparison of the anticancer ability of different doses and whether various doses can lead to stable proteinuria have not been reported. Our results showed that in both liver cancer animal models, 200 mg/kg/day apatinib exhibited the best anticancer effect, prolonged the survival of tumor-bearing mice (Figure 2, b and f) and inhibited tumor growth (Figure 2, c and g). Importantly, the 200 mg/kg/day apatinib group produced stable proteinuria after two weeks (Figure 2, d and h). Targeting VEGF may cause two types of glomerular injury: thrombotic microangiopathy (TMA) and focal segmental glomerulopathy (FSGS) [42]. Both of them were observed in our apatinib-induced proteinuria mouse model (Figure 2i). These data con rmed that the optimal dose of apatinib was selected when studying the effect of apatinib on proteinuria.
Captopril does not reduce proteinuria or kidney damage caused by apatinib In clinical practice, cancer patients may use ACEIs after the diagnosis of proteinuria caused by AADs. To t the clinical situation, we administered apatinib to tumor-bearing mice for 2 weeks in advance to ensure stable proteinuria and then administered captopril to determine whether captopril could alleviate proteinuria (Figure 3a). From the initiation of captopril administration, the 24-hour urine of mice was collected via metabolic cages each week. From the weekly dynamic quantitative test and the qualitative test of the last week of captopril administration, the administration of captopril was not found to effectively alleviate albuminuria (Figure 3, b and c). Histological analysis of HE-stained, PAS-stained and Masson-stained kidney samples showed that captopril treatment neither attenuated the degree of glomerular injury nor reduced the proportion of damaged glomeruli (Figure 3, d and e). CD31 immuno uorescence staining of the kidneys showed a signi cant decrease in microblood vessels after apatinib treatment, which was not attenuated by captopril treatment (Figure 3f). Furthermore, captopril did not interfere with the effect of apatinib on the molecular level of the renal VEGF signaling pathway (Figure 3g). These data con rmed that captopril cannot reduce the proteinuria or kidney damage caused by apatinib.
ACEIs do not delay proteinuria or kidney damage caused by AADs Although ACEIs have a de nite effect on delaying proteinuria in diabetic nephropathy, ACEIs had no signi cant effect on the event of death [43]. Next, we sought to observe whether ACEIs could delay the appearance of proteinuria caused by AADs. We simultaneously administered different ACEIs and AADs to tumor-bearing mice (Figure 4a, Supplemental Figure 4a). Because Regorafenib has been shown to induce a higher rate of proteinuria when used to treat liver cancer [44], we selected apatinib and regorafenib as representatives of AADs, and we selected captopril, enalapril and fosinopril as representatives of ACEIs.
Our data showed that although the combined use of ACEIs and AADs in the rst three weeks resulted in lower quanti ed proteinuria than AADs alone, the difference was not statistically signi cant. After proteinuria was stabilized, proteinuria under treatment with ACEIs and AADs was almost the same as proteinuria induced by AADs alone (Figure 4b, Supplemental Figure 4c). Qualitative proteinuria in the urine of mice in the last week also con rmed that the combination of ACEIs could not reduce the  Figure 4h). Together, the above results indicated that ACEIs could not delay the occurrence of proteinuria end events caused by AADs.
ACEIs do not affect the reduction in proteinuria or the recovery of kidney injury after AAD withdrawal As shown in Figure 1a, we found that after the use of AADs was stopped in patients, proteinuria gradually decreased and disappeared. Administration of the drugs was stopped after three weeks of treatment with apatinib and captopril, and the proteinuria of the mice was tested every week after administration ( Figure  5a). We found that the proteinuria of the mice gradually disappeared after three weeks of apatinib withdrawal (Figure 5b). Pathological analysis of the kidneys revealed that glomerular damage was alleviated, and the proportion of damaged glomeruli was reduced from approximately 80% to 25% ( Figure   4c and Figure 5d). Together, these results suggested that ACEIs do not delay or reduce the proteinuria caused by AADs. However, proteinuria gradually disappears, and kidney damage recovers after AAD withdrawal.
ACEIs reduce the anticancer e cacy of AADs, and their combined treatment promotes liver and lung metastasis in tumor-bearing mice with high metastatic potential We tested three kinds of ACEIs and two kinds of AADs in two animal models to observe whether ACEIs could reduce the anticancer effect of AADs in different drug combinations. In tumor-bearing immunode cient mice with MHCC-97H cells, we found that the use of low-dose ACEIs alone affected neither survival nor tumor growth. The combination of ACEIs and AADs inhibited tumor growth in tumor-bearing mice and improved survival. However, the improvement observed for the combined use was signi cantly lower than that observed for the use of AADs alone (Figure 6, a, b, d and e). We also measured the volume and mass of the tumors at speci c time points. The combined use of ACEIs and AADs still corresponded to signi cantly higher tumor volume and mass than the use of AADs alone ( Figure 6, c and f). We also observed liver and lung metastasis in mice. In the combined treatment group, some mice with subcutaneous tumors were found to have metastasis to the liver and lungs, in the ACEIs alone or AADs alone group, no liver or lung metastases were observed (Figure 6g).
In tumor-bearing immunogenic mice with Hep1-6 cells, low-dose ACEIs did not affect tumor growth or reduce the survival time. Although the combined use of ACEIs and AADs improved survival and inhibited tumor growth, the effects were signi cantly lower than those of AADs alone (Supplemental Figure 5). Consistent with the clinical observations shown in Figure 1 and Supplemental Figure 1, ACEIs did have an effect on reducing the anticancer e cacy of AADs, and the combined treatment of ACEIs and AADs may also promote the metastasis of cancer cells.

ACEIs reduce the anticancer e cacy of AADs by promoting the expression of kidney-derived EPO
In the process of exploring the mechanism by which ACEIs reduce the anticancer e cacy of AADs, a phenomenon that has attracted our attention, we noticed that the capillaries on the surface of the subcutaneous tumor were almost invisible in the nude mice treated with apatinib. However, after combined treatment with captopril, the capillaries on the surface of the tumor reappeared (Supplemental Figure 6a). We doubt whether the combined use of ACEIs reduces the anti-angiogenic e cacy of AADs. CD31 immuno uorescence staining revealed that, unlike in the kidney, ACEIs reduced the antiangiogenesis effect of AADs in the tumor mass (Figure 7a, Supplemental Figure 6b). We further explored the effect on the VEGF signaling pathway in the tumor mass. As in the kidney, ACEIs did not interfere with the inhibition of VEGF signaling by AADs (Figure 7b, Supplemental Figure 6c). Because activation of the EPO signaling pathway has been well proven to be a main reason for the off-target anti-angiogenesis effect of AADs [21,23], we analyzed the EPO signal of tumor tissues. The EPO and EphB4 protein levels in tumor tissues of the ACEI combination group were signi cantly higher than those of the AADs alone group (Figure 7c, Supplemental Figure 6d). The trend of EPO levels in the mouse serum was consistent with that in the tumor tissue (Figure 7d, Supplemental Figure 6e).
The liver, kidney, spleen and tumor are potential organs or tissues for EPO synthesis [21,27]. Therefore, EPO mRNA was measured in these tissues, and no signi cant change was observed in the liver, spleen or tumor tissues. However, a signi cant increase in EPO mRNA was detected in the kidney in the ACEI combination group (Figure 7e, Supplemental Figure 6f). Western blotting and immunohistochemical staining further con rmed that the combination of ACEIs increased the levels of protein related to the EPO pathway to a greater degree than the use of AADs alone without affecting the VEGF signaling pathway (Figure 7f and 7g, Supplemental Figure 6g). These ndings indicate that ACEIs aggravate the production of renal EPO caused by AADs, which leads to the rapid emergence of AAD resistance.

Discussion
Our data support the following main ndings. (a) ACEIs cannot reduce or delay proteinuria caused by AADs, (b) ACEIs alone do not promote tumor growth of immune integrity and immunode ciency liver cancer subcutaneous tumor mouse model. (c) High doses of apatinib easily induce stable proteinuria, but the anticancer effect of apatinib is better. (d) Proteinuria caused by AADs is reversible after withdrawal. (e) The combination of ACEIs and AADs promotes the production of kidney-derived EPO and reduces the anticancer e cacy of AADs. In summary, ACEIs cannot relieve AAD-induced proteinuria but promote drug resistance during the treatment of hepatocellular carcinoma.
Maintenance of the physiological function of the glomerular ltration barrier requires ne crosstalk between VEGF on podocytes and VEGFR2 on endothelial cells [45,46]. VEGF levels that are too high or too low can both lead to proteinuria [47,48]. Proteinuria caused by diabetic nephropathy has been shown to be closely related to activation of the VEGF signaling pathway, and the pharmacological inhibition of VEGF helps reduce diabetic nephropathy-related proteinuria [49]. The proteinuria caused by AADs is an effect of inhibition of the VEGF signaling pathway [8], therefore, considering the VEGF signaling pathway, the mechanism of the occurrence of proteinuria is opposite. Some researchers believe that ACEIs delay proteinuria in diabetic nephropathy due to inhibition of the VEGF signaling pathway [50]. Our results indicate that ACEIs alone or in combination with AADs do not affect the kidney's VEGF signal (Figure 4 and 5). According to our experimental results, ACEIs are unlikely to reduce proteinuria caused by AADs. As shown in Figure 4b and Supplemental Figure 4c, the combination of ACEIs at the initial stage slightly reduced proteinuria, but the difference was not signi cant. Rather, we speculate that the effect of ACEIs on hemodynamics reduces renal perfusion and may have a certain effect on proteinuria in the initial stage, although this effect is slight and time-limited. Unfortunately, our ndings indicate that ACEIs have no e cacy in reducing or delaying proteinuria caused by AADs.
It has been reported that proteinuria caused by AADs can be restored after drug withdrawal. We veri ed this point in our tumor-bearing mouse model. The reduction in proteinuria is inseparable from reactivation of the renal VEGF signaling pathway, angiogenesis, repair of glomerular injury and functional recovery. However, discontinuation also means halting the anti-angiogenic therapy against the tumor. It is well believed that the counterattack of tumor angiogenesis is also rapid and that the tumor will rapidly progress after drug withdrawal. Many oncologists believe that drug withdrawal should only be implemented with caution. The optimal management strategy for increasing or decreasing AADs and drug withdrawal requires further evidence-based research. AAD-induced nephrotoxicity versus anticancer e cacy seems to be an irreconcilable paradox. However, a considerable number of patients still receive AADs in the clinic who do not develop proteinuria, which may be related to the patient's basic renal function and the tolerance of the kidneys to AADs. Further investigations are needed to determine how to reduce or reverse proteinuria without compromising the antitumor effect of AADs.
The results of epidemiological studies on the relationship between ACEIs and malignant tumors are controversialand this controversy has been driven by insu cient follow-up, regardless of cancer type and imperfect methodology [15,17,51]. Higher quality research has gradually emerged in recent years. For example, Hicks et al. con rmed that the long-term use of ACEIs for more than 5 years clearly promotes lung cancer [18,52]. The effects of ACEIs in tumor-bearing mice also differ across different tumor types, different tumor-bearing mouse models and different drug dosages. Similar to the study by Piotr J et al., we focused on the effects of well-tolerated doses of ACEIs on tumor growth in mice [19]. Our results show that regardless of immunogenic or immunode cient tumor-bearing mice, ACEIs alone do not affect tumor growth. This result may be strongly related to the mouse model. Our research is consistent with the effect of ACEIs on subcutaneous tumors con rmed by other studies [53,54].
It has been reported that angiotensin system inhibitors can improve vessel perfusion and promote nanomedicine or chemotherapy drug delivery, thereby improving the e cacy of apoptotic drugs [53,54]. The effect of angiotensin system inhibitors on AADs has not been well elucidated, and here, we rule out immune interference to prove that ACEIs reduce the anticancer e cacy of AADs against liver cancer. Supplemental Figure 6a shows the conversion of AAD-treated subcutaneous tumors from white to red upon administration of ACEIs. As shown in Supplemental Figure 6f, the experimental tumors treated with ACEIs were more prone to intratumoral hemorrhage. These phenomena are all related to the fact that ACEIs promote tumor blood vessel perfusion. Importantly, CD31 staining clearly showed that ACEIs reduce the anti-angiogenic effects of AADs. Therefore, we proved that ACEIs compromise the anticancer activity of AADs.
We performed a preliminary investigation to further delineate why ACEIs reduce the anticancer e cacy of AADs. ACEIs do not affect the VEGF signaling pathway of tumors but promote the production of renal EPO caused by AADs. Renal-derived EPO may then act on the tumor site to cause AAD resistance.
Because our data con rmed that physiologically tolerable doses of ACEIs are ineffective against proteinuria caused by AADs and that they promote the development of AAD resistance, we believe that the combined use of ACEIs in cancer patients who are being treated with AADs should be carefully reconsidered. Taken together, ACEIs have no e cacy for the treatment of proteinuria caused by AADs, but promote drug resistance. Kidney-derived EPO is mainly responsible for ACEIs induced anti-angiogenesis resistance. Most importantly, ACEIs should be used very cautiously in patients with anti-angiogenic therapy.

Conclusion
Taken together, our data show that systemic anti-angiogenesis treatment leads to a reduction in tumor blood vessels and reduces kidney blood vessels, which is the root cause of kidney damage and proteinuria. ACEIs cannot reduce the damage of AADs to the kidneys but increase the production of EPO induced by AADs, which indirectly accelerates the resistance of tumors to AADs (Figure 8). This study is of great signi cance to guide the use of ACEIs in the process of antiangiogenic therapy in patients with hepatocellular carcinoma.    = 3 samples per group). (e) Kidney CD31+ staining of the mice. CD31+ staining was randomly quanti ed from 12 elds (n = 6 samples per group). *p<0.01, **p<0.001, ns represents no signi cant difference. Percentage of damaged glomeruli in each pathological section. *p<0.01, ns represents no signi cant difference. *p<0.01, **p<0.001, ns represents no signi cant difference.

Figure 6
ACEIs abate the anticancer ability of AADs in tumor-bearing immunode cient mice and promote liver and lung metastasis. Abbreviations: apatinib + captopril (A+C), apatinib + enalapril (A+E), apatinib + fosinopril (A+F) and regofenib + captopril (R+C). (a, d) Survival curves of subcutaneous tumor-bearing mice treated with ACEIs alone, AADs alone, or a combination of both (n = 8). (b, e) Measurement of tumor volume at different time points after ACEIs alone, AADs alone, or a combination of both administration (n = 8). (c, f) Subcutaneous tumors and quanti cation of their mass and volume at certain time points (n =6, 7 or 8).
(g) Representative images of HE-stained sections of the liver and lungs for subcutaneous tumor metastasis to the liver and lung and the number of mice with liver and lung metastasis. *p<0.01, **p<0.001, ns represents no signi cant difference. Figure 7