Changes in PD-L1 expression on microvessel endothelial cells during melanoma tumor growth and the regulation by anlotinib

Changes in PD-L1 expression on microvessel endothelial cells during melanoma tumor growth and the regulation by anlotinib | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Changes in PD-L1 expression on microvessel endothelial cells during melanoma tumor growth and the regulation by anlotinib Yingfang Feng, Yuan Gao, Tingting Qin, Yan Zhang, Shaochuan Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3231581/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To evaluate the expression of PD-L1 on MEC in tumor and normal tissues, IF was used to detect the expression of PD-L1 + CD31 + VECs and PD-L1 + Podoplanin + LECs in tumor, ear and kidney tissues. We found that the MEC PD-L1 in tumor showed an upward trend with tumor progression. The expression of PD-L1 in MEC in normal tissues is associated with the type of microvessels and tumor progression. The MEC PD-L1 expression in tumor is significantly down-regulated at 22th day in An5-9 group. In the early treatment group (An5-9), anlotinib increased the LEC PD-L1 but had no significant effect on VEC PD-L1 of ear, while decreased the LEC PD-L1 in kidney. In conclusion, the MEC-PD-L1 in melanoma increased with tumor growth and was downregulated significantly by early treatment of anlotinib, which proposes a potentiality of its enhancement on the therapeutic efficacy of anti-PD-L1 antibody. Anlotinib had no significant effect on the MEC PD-L1 of ear excepting the upregulation of LEC PD-L1 in early treatment group, while decreased such expression in kidney, hence the risk of possible renal damage by CD8 + T cells in the treatment of anlotinib should be bewared. vascular endothelial cells (VEC) lymphatic endothelial cells (LEC) PD-L1 melanoma Anlotinib Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction PD-1 is an immunosuppressive receptor expressed on the surface of activated T cells, B cells and bone marrow cells( 1 ); and it’s ligand, PD-L1, is expressed on a variety of cells, including macrophages, dendritic cells, tumor cells, lymphocytes in draining lymph nodes, and non-lymphoid tissues (such as heart, lung)( 2 , 3 ). The binding of PD-L1 on tumor cell to PD-1 on T cell inhibits the activation and proliferation of T cells, leading to the depletion of CD8 + T cells, inducing tumor tolerance to T cells and promoting tumor immune escape( 4 ). So far, the majority of research has focused on the connection between the high PD-L1 expression on tumor cells and the ideal efficacy of anti-PD-L1. However, still there are a considerable number of patients whose tumor cells with high expression of PD-L1 revealed poor therapeutic efficacy of anti-PD-L1, suggesting that there are other mechanisms affecting the prognosis beyond the PD-L1 on tumor cells( 5 ). Recent researches proved that PD-L1 on vascular endothelial cells (VEC) and lymphatic endothelial cells (LEC) could be induced by tumor and banded with PD-1 on CD8 + T lymphocytes, inhibiting T cell infiltration and leading to its depletion( 3 , 6 , 7 ), hence leading to the escape of tumor immune damage. Moreover, those PD-L1 on tumor-associated micro-vascular/lymphatic endothelial cells (MEC) could “consume” and waste the PD-L1 antibody that should be banded with the PD-L1 on tumor cells. Therefore, how to remove the inhibition on CD8 + T cells by PD-L1 on the surface of tumor-associated MEC to break this "immune barrier" has become another key point to improve the efficacy of immunotherapy. Anlotinib, a small molecule multi-targeted receptor tyrosine kinase inhibitor, which inhibited VEGFR1-3, FGFR1-4, PDGFR α and β, and C-kit, can inhibit tumor cell proliferation and anti-tumor angiogenesis( 8 , 9 ). Our previous studies have shown that anlotinib could inhibit tumor growth by down-regulating the expression of PD-L1 on tumor vascular endothelial cells to improve the infiltration and activity of lymphocytes in tumor tissues( 10 ), which provides its potential application in synergy with immunotherapy. However, the changes of PD-L1 on VEC and LEC in tumor and normal tissues during tumor growth have not been dynamically observed, neither the effects of anlotinib on the expression of PD-L1 on MEC in tumor and normal tissues has been observed. This study aims to observe the above indexes and evaluate the optimal time of administration of anlotinib to get the best synergistic effect with immunotherapy, and potential damage by infiltrative CD8 + cells through MEC into normal tissues, for providing the reference to clinical treatment. Materials and methods Cells culture and reagents B16 cells was obtained from Tianjin Medical University Cancer Institute and Hospital. Female C57BL/6J mice aged 6–8 weeks were purchased from the SPF (Beijing) Biotechnology Co.,Ltd. All the experimental program conforms to the tumor hospital of tianjin medical university laboratory animal ethics rules. Anlotinib was provided as gift by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. The cells were cultured with DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a humidified incubator containing 5% CO2. In vivo experiment To construct a tumor-bearing mouse model of B16 cells, we injected 1× 10 6 /100 µ L B16 cells subcutaneously into the right groin region of each mouse. Tumor growth was observed every day for several weeks. Based on the previous studies, tumor volume was calculated by the formula of volume = length × width × width /2( 11 ), and the 22nd day was set as the end time of mice’s survival. The tumor growth curve was drawn, and B16 melanoma was divided into three growth stages according to the tumor volume growth rate( 12 , 13 ), approximately simulating the clinical T stage, namely day5-9, day 10–14, and day16-20. In order to explore when the administration of anlotinib has an optimal effect, we established B16 xenograft tumor model and divided mice into four groups, NC (PBS, 1.5 mg/kg, once daily), An5-9 (anlotinib, 1.5 mg/kg, once daily for day5-9), An10-14 (anlotinib, 1.5 mg/kg, once daily for day10-14) and An16-20 (anlotinib, 1.5 mg/kg, day16-20 once daily), respectively. We compared the changes of MEC PD-L1 in normal mice and non-drug-treated tumor-bearing mice at the 10th, 15th and 22nd days as observation points. For mice in each treatment group, the 22nd day after tumor bearing was selected as the observation point. Selection of tissue (organs) to observe the PD-L1 on MEC According to the previous research, characteristics of PD-L1 expressed by VEC and LEC are different, which suggested that tumor might have different effects on them, so VEC and LEC were selected respectively to be observed. Tumor and normal tissue can both be influenced by medicine, so we choose both of them. In the common view of the organs with side effects of immunotherapy, we choose ear tissue representing the peripheral skin and kidney tissue representing the viscera. Immunofluorescence staining (IF) The fresh frozen sections were rewarmed at room temperature for 10 minutes, then the tissues were fixed with 4% paraformaldehyde precooled 4℃ for 15 minutes. The frozen tissue samples after fixation were permeated with 0.2% TritonX-100 diluted with PBS for 10 minutes. Washing with PBS 3 times, for 5 minutes each time, and all sections were incubated with PBS block solution containing 1% BSA, 0.01% Triton X-100 and 10% FBS at room temperature for 1h. The slices were incubated with primary antibody overnight at 4℃. The primary antibodies used were as follows: Rat anti-mouse CD31(AB56299, Abcam), Syrian hamster anti-mouse Podoplanin (AB11936, Abcam), rabbit anti-mouse PD-L1 (LS-C746930, LifeSpan). The sections were then rewarmed at room temperature for 30 min and washed in PBS 3 times, 5 min each. The tissue sections were stained with fluorescent secondary antibody and incubated at room temperature for 1h. The following secondary antibodies were used: donkey anti-rat AF488 (A21208, Invitrogen), donkey anti-Syrian hamster AF546 (A21111, Invitrogen), donkey anti-rabbit AF647 (A31573, Invitrogen); then washed in PBS for 3 times. Finally, all the sections were stained with anti-fluorescence quencher (including DAPI). The stained sections were stored at -20℃, protected from light, and Zeiss Imaginer-Z2 was used for image acquisition. Evaluation the percentage of PD-L1 + MEC in tissues CD31( 14 ) and Podoplanin( 15 )was used to label vascular endothelial cells and lymphatic endothelial cells, respectively. To assess the percentage of CD31 + PD-L1 + and Podoplanin + PD-L1 + in tissues, the sites with the most abundant blood vessels and lymphatic vessels were selected under 10-fold microscope, 3–5 independent fields were randomly selected under 40-fold objective lens, and the percentage of CD31 + PD-L1 + cells in all CD31 + cells and Podoplanin + PD-L1 + cells in all Podoplanin + cells was calculated. Take the average and use it as the final result for each section( 10 , 16 ). Statistics Statistical analyses were calculated using SPSS v.24 (IBM Corp), statistical graphs and analyses were generated using GraphPad Prism 8 (USA, GraphPad Software). All measurement data were expressed as mean ± standard deviation, unpaired T test was used for comparison between two groups of data, and one-way ANOVA was used for comparison between multiple groups of data. Wilcoxon rank sum test was used for non-normally distributed data. P < 0.05 was considered to be statistically significant. Results Expression of PD-L1 on VEC and LEC of normal tissues in naive mice Immunofluorescence staining showed that VEC PD-L1, namely CD31 + PD-L1 + /CD31 + (%) expression was 68.41±18.44 in the ear tissues of non-tumor burden mice. The expression of LEC-PD-L1, namely Podoplanin + PD-L1 + / Podoplanin + (%), was 33.92±20.17 (Fig.1). The positive expression rate of VEC-PD-L1 and LEC-PD-L1 in kidney tissues of non-tumor burden mice was 14.25±5.864 and 24.16±17.75 (Fig.2). PD-L1 expression of VEC and LEC in ear tissues of tumor-bearing mice during tumor growth CD31 + PD-L1 + /CD31+(%) in PBS group were 16.10±5.933, 16.13±9.228 and 12.63±5.444 at day 10, 15 and 22, respectively. Compared with normal mice, tumor-bearing mice VEC PD-L1 expression was significantly decreased (P<0.0001) (Fig.1A, C). Podoplanin + PD-L1 + / Podoplanin + (%) in PBS group on day 10, 15 and 22 were 17.09±3.657, 18.04±12.44 and 17.75±11.39, respectively. The expression of LEC-PD-L1 declined on day 10 and 22 compared with ear of normal mice (P=0.0248, P=0.0295, respectively) and there was no significant difference in the expression of LEC-PD-L1 on day 15 in tumor-bearing mice compared with normal mice (P>0.05) (Fig.1B, D). PD-L1 expression of VEC and LEC in renal tissues of B16 mice during tumor growth CD31 + PD-L1 + /CD31 + (%) in PBS group were 31.52±12.52, 27.91±14.45 and 14.48±3.930 on day 10, 15 and 22, respectively. Compared with vec-PD-L1 in renal tissues of normal mice on day 0, there was no significant difference in VEC-PD-L1 expression after tumor bearing (P>0.05) (Fig.2A, C). PODOPLANIN + PD-L1 + /PODOPLANIN + (%) in PBS group were 23.28±8.071, 25.70±14.15 and 35.87±14.63 at day 10, 15 and 22, respectively. Compared with day 0 (normal mice), LEC-PD-L1 in renal tissue tended to be up-regulated after tumor loading, with a near significant difference at day 22 (P=0.2408) (Fig. 2B, D). VEC and LEC expressed PD-L1 in B16 tumors with tumor growth According to previous literature, both VEC and LEC of tumor tissues express PD-L1, which is induced by IFN-γ that produced by tissue-infiltrating, antigen-specific CD8 + T cells(14). However, little research on the dynamic changes of MEC PD-L1 with tumor growth, so the expression of CD31 + PD-L1 + /CD31 + (%) and PODOPLANIN + PD-L1 + / PODOPLANIN + (%) were compared at days 10, 15 and 22 after tumor cells were inoculated. We found that with tumor progression, VEC PD-L1 was escalate. Compared with the quantitative value on day 10, VEC PD-L1 on day15 was not significantly up-regulated (P=0.3899), while that on day22 was significantly up-regulated (***P= 0.0005) (Fig. 3A, C). LEC PD-L1 was also increased in tumor tissues, and compared with the percentage of day10, it was not significantly up-regulated at day15 (P=0.9944), while significantly up-regulated at day22 (**P=0.0024). Tumor-associated MEC PD-L1 were increased across the progression of tumor (Fig. 3B, D). Effects of anlotinib given at different stages of tumor on PD-L1 expression of VEC and LEC in ear tissues Compared with the control group, the proportion of CD31 + PD-L1 + /CD31 + (%) was slightly up-regulated in all treatment groups, without statistical significance (P>0.05) (Fig. 4A, C). In Comparison with the control group, the expression of Podoplanin + PD-L1 + / Podoplanin + (%) was significantly up-regulated in the early treatment group (**P=0.0019) (Fig. 4B, D). It was slightly up-regulated in the middle dosing group, and slightly down-regulated in the late dosing group, with no statistical significance (P>0.05). Effects of anlotinib given at different stages of tumor on PD-L1 expression of VEC and LEC in renal tissues In comparison with the control, the rate of CD31 + PD-L1 + /CD31 + (%) was slightly upregulated in the treatment groups, without statistical significance (P>0.05) (Fig.5A, C). While Podoplanin + PD-L1 + / Podoplanin + (%) was down-regulated in all treatment groups compared with the control group, (Fig.5B, D), and there were statistically significant differences in the early and middle dose-adding groups (P=0.0232, P=0.0037). There was no statistical significance in late treatment group (P>0.05). Effects of anlotinib given at different stages of tumor on PD-L1 expression of VEC and LEC in tumor tissues Our previous study found that anlotinib could attenuate the expression of PD-L1 in microvascular endothelial cells. Compared with the day22 in control group, VEC-PD-L1 was down-regulated in each period after administration, and the differences were statistically significant (****P<0.0001, ***P=0.0002, **P=0.0097). And the most obvious down-regulation was observed at early treatment group (Fig.6A, C). In comparison with the control group, LEC-PD-L1 was down-regulated in each period in treatment group with the significant differences (****P<0.0001, ****P<0.0001, ****P<0.0001). There was no statistically difference between treatment groups at each stage (Fig.6B, D). Discussion Regulation of PD-L1 in endothelial cells The application of immune checkpoint inhibitors (ICIs) have promoted the treatment of cancer. ICIs targeting programmed death ligand 1 (PD-L1) have shown striking anti-tumor efficacy in a variety of cancers(17). While only a part of patients benefit from it in clinical implementation(18-21). This may be related to the fact that MEC can also express PD-L1 and form an "immune barrier" to prevent active lymphocytes from infiltrating into tumors. Conversely, down-regulation of PD-L1 expression on MEC could weaken this barrier and promote CD8 + Teff cells to infiltrate the tumor and improve the immunotherapy effect(10). In MECs, recent study(14) has found that LEC and VEC can express PD-L1 in melanoma and inflammatory skin, VEC can express PD-L1 systemically in all conditions, regardless of whether or not the tumor is carried and what the type of tumor, while LEC demonstrated high specificity to the local microenvironments, even high expression of LEC-PD-L1 in normal tissues only on the ipsilateral side of the lesion (" near area "), but low expression of LEC-PD-L1 in normal tissues on the contralateral side of the lesion (" far area ") were found(14). The authors believe that it was due to the different properties of the two types of MEC. LEC is more likely to show an "active response" to surrounding lesions than the "passive" of VEC. In addition, the supply of blood and oxygen in different tissues are also important factors that affect the expression of PD-L1 in MEC. The expression of PD-L1 is regulated by a variety of mechanisms, the following are elaborated separately. (1) CD8 + T cells and secreted factors in local tissues The accumulation of antigen-specific T cells and the activation of local TCR increased the concentration of IFN-γ in the infiltrated tissues, and IFN-γ activated LEC by inducing PD-L1 expression through the JAK/STAT pathway; subsequently, LEC-PD-L1 limited the infiltration of activated CD8 + T cell and protected its "targeted tissue" in turn. Several studies have confirmed that endogenous expression of interferon regulatory factor (IRF-1) is necessary for constitutive and inducible B7-H1 transcription and regulates IFN-γ induced B7-H1 (CD274, PD-L1) through the JAK/STAT pathway(22, 23). Anti-angiogenic drugs could facilitate the accumulation of perivascular activated CD8 + T cells to up-regulate the expression of endothelial PD-L1 through IFN-γ(24). It should be noted that such a regulatory effect occurs and exists only between T cells and their adjacent LEC, and their spatial distribution must be very coincidence. However, the regulatory effect of T cells on distant LEC is not strong as well as weaker on VEC(14). Therefore, this effect mainly regulates the local and peripheral tissues near the lesion(14). In this study, we observed that consistent with the trend in tumors, the LEC PD-L1 of kidney in B16 melanoma model was increased at day 22 of tumor growth, closing to a statistically significance. So, we speculated that this pathway mainly regulates the expression of PD-L1 in LEC of tumor, and kidney tissue that easier obtain the regulators form tumor through the rich of blood perfusion (equivalent to "near tumor tissues") so that more CD8 + T cells could infiltrate into tissue to secrete IFN-γ. However, LEC PD-L1 may not be mainly regulated by this mechanism in ear tissues (equivalent to "distal tumor tissues") that are not rich in blood flow and receive too little chemokines (such as CCL4, CCL5 and CXCL9(25, 26) to recruit CD8 + T cells, neither may be regulated by such way in VEC with “passive response” to local immune environment. (2) HIF-1 α and VEGF-NO pathway After post-translational modification of HIF-1α (hypoxia-inducible factor 1α subunit) by prolyl hydroxylase under normoxic conditions, the hydroxylase binds to pVHL (von Hippel-Lindau protein) to induce ubiquitination of HIF-1α(27). Hypoxia is common in tumors and some relatively ischemic tissue (organ), in this way, the binding of pVHL to HIF-1α and ubiquitin degradation is attenuated, which results in the accumulation of HIF-1α. Simultaneously, it forms a dimer with HIF-1β, which is subsequently transferred to the nucleus(28), where HIF-1α binds to a transcriptionally active hypoxia response element (HRE) in the proximal promoter of PD-L1, leading to activated transcription of PD-L1(29). VEGF produced by tumor tissues can upregulate eNOS (endothelial nitric oxide synthase) in endothelial cells to elicit the release of NO by endothelial cells and loosen smooth muscle, resulting in microvasodilation and increased vascular perfusion(30-34), relieving hypoxia in near and distant tissues. (3) PI3K-AKT pathway Activated PI3K/AKT pathway can up-regulate PD-L1 expression on vascular endothelial cells (10, 35). This pathway mainly plays a role in local tumor tissue. Regulation of dominance in different tissues The expression of VEC PD-L1 in tumor tissues increased with tumor growth, the regulatory mechanism could be the following: (1) local lesions oxygenation decreased, the hypoxia-HIF-1α-PD-L1 pathway was active; (2) the secretion of VEGF increased in tumor tissues, which could activate VEGF-PI3K/ AKT-PD-L1 pathway; (3) the IFN-γ-JAK /STST-PD-L1 pathway, which was related with CD8 + T cells. Similar to VEC, LEC-PD-L1 in tumor tissues was also enhanced with the mechanism described above. The expression of PD-L1 in MEC of ear tissue in tumor-bearing mice was significantly down-regulated compared with normal mice, especially in VEC. This can be attributed to the fact that ear tissue is a terminal circulating tissue and in a state of relative hypoxia. Thus, the regulatory pathway of HIF-1α played a dominant role in regulation, with high expression of PD-L1 in MEC. However, when amounts of VEGF was secreted by carrying tumors, for the ear tissue of poor terminal microcirculation, their relatively contracted micrangium may be expanded by VEGF-eNOS-NO regulation more than the vessels in blood-rich, oxygenated renal tissue. So, in the case of ear, the control effect of vascular vessel is also stronger than lymph vessels; the expression of VEC PD-L1 was decreased in ear but not in renal tissue in tumor-burdened mice. VEC PD-L1 expressed without significant change in renal tissues between normal and tumor-bearing mice. As in normal mice, renal tissue has adequate blood perfusion and oxygenation with low HIF-1α; while in tumor-bearing mice, oxygen supply could be increased only slightly, vasodilatation by VEGF-eNOS-NO pathway was limited, so HIF-1α was not significantly decreased. Meanwhile, the expression of LEC PD-L1 was up-regulated in renal tissues (especially at the 22d of advanced tumor), the reason could be numerous lymphocytes activating factors produced by tumors at this time to attract lymphocytes into renal tissues (as a blood-rich “near tumor tissue”) and secrete IFN-γ to activate the JAK/STAT pathway in renal LEC to up-regulate the expression of PD-L1. Meanwhile, NO had a better impact on vascular dilation than on lymph vessel. The IFN-γ-JAK /STST-PD-L1 pathway activated by CD8 + T cells played the major role, promoting the upregulation of LEC-PD-L1. Regulation of anlotinib in different tissues Anlotinib targets VEGFR2, PDGFRβ and FGFR1(36), and our previous studies have demonstrated that anlotinib can down-regulate PD-L1 expression in VEC through inactivation of Akt(10). Other studies have also confirmed that anlotinib can inhibit the activation of PI3K/Akt pathway(37, 38). In addition, several studies have found that anlotinib can inhibit JAK2/STAT3/VEGFA signaling pathway(39, 40). However, the distribution of anlotinib is significantly affected by the status of blood perfusion. The expression of VEC-PD-L1 was slightly upregulated in ear tissues of each treatment group in tumor-bearing mice. This is probably because, on the one hand, anlotinib targets VEGFR so the VEGF-eNOS-NO pathway was limited, ear vessels return to the state of relative hypoxia; on the other hand, as a small-molecule drug, a little anlotinib may reach to VEC and reduce the expression of VEC-PD-L1 by inhibiting the PI3K/AKT pathway. The ear LEC-PD-L1 was significantly upregulated in the early treatment group, while was slightly down-regulated in the late treatment group, which may be due to the inhibition of VEGF-NO pathway in early stage of tumor to form more obvious impact as aforementioned on more ischemic LECs. When the tumor grew to advanced stage, anlotinib could not completely inhibit the impact of VEGF, the ear lymphatic vessels were relatively dilated, and HIF-1α was degraded. Therefore, the expression of LEC-PD-L1 in the advanced treatment group was slightly lower than that in the control group. The expression of VEC-PD-L1 in renal tissues was slightly upregulated in each treatment group. We speculated that anlotinib inhibited the expression of VEC-PD-L1 by inhibiting PI3K/AKT pathway, but inhibited VEGFR to weaken the VEGF-eNOS-NO pathway, so activated HIF-1α-PD-L1 pathway. The two effects interact with each other so that VEC-PD-L1 kept approximately stable. The renal LEC-PD-L1 was down-regulated in treatment groups, which might be attributed to the fact that anlotinib could act on the renal lymphatic vessels and inhibit the activated PI3K/AKT and IFN-γ-JAK/STAT pathway. Although anlotinib also inhibits lymphatic vessel expansion by inhibiting VEGFR, leading to hypoxia and upregulating HIF-1α, it was obviously weaker than the same impact on VEC. In tumor tissues, the VEC-PD-L1 declined in anlotinib groups. Based on the results of previous studies, we analyzed that anlotinib inhibited PI3K/AKT pathway and improved hypoxia to decrease HIF-1α by inhibiting VEGF. LEC-PD-L1 in tumor tissue was also down-regulated for the similar mechanisms and the inhibition of JAK/STAT signaling. Early treatment of anlotinib may enhance the therapeutic efficacy of PD-L1 antibody In present study, the expression of PD-L1 on tumor tissues VEC and LEC was upregulated with tumor progression, so both of the expression of PD-L1 on VEC and LEC can constitute an "immunosuppressive barrier" of tumor like our previous results (10). Anlotinib can significantly down-regulate PD-L1 of MEC when administered at the early stage of tumor growth, which may help to remove the immune barrier formed by microvessels expressed PD-L1 in tumor microenvironment, promote more Teff cells to infiltrate tumor tissues out of blood and lymphatic vessels.In this way, its combination with anti-PD-1 / anti-PD-L1 drugs can achieve optimal therapeutic effect. Potential damage of anlotinib on different organs in immunotherapy Our previous study showed that the down-regulation of PD-L1 expression on VEC by anlotinib can accelerate the infiltration of CD8cells in tissues and contribute to tumor control. However, the more immune cells infiltrate in normal tissue, the more attacks could happen. According to the dynamic change of PD-L1 expression in MEC of ear and kidney tissues in different groups, compared with control group, LEC-PD-L1 in ear was significantly up-regulated in the early administration group, while there was no significant difference in middle and late administration group. Therefore, we speculated that anlotinib administration at different stages may not cause severe immune "toxification" in peripheral tissues (distal tumor tissues, such as ear skin). However, LEC-PD-L1 in kidney was down-regulated. Therefore, it is necessary to be vigilant that drug administration may cause immunotoxicity to renal tissues to some degree. In summary, in present study, we elucidated the changing trend in PD-L1 expression on MECs in tumor and normal tissue, and the effect of anlotinib on the various tissue (organ)s during the tumor growth. We believe our discovery should be valuable and referential for determining the optimal administrative time of “immuno-efficacy-accelerating agent” of anlotinib to cut down the “immune barrier” of PD-L1 on MEC (especial on LEC) to reinforce the PD-L1 antibody, and avoiding the toxicity of PD-L1 antibody in normal tissue. Limitation of this study This study only observed the expression of PD-L1 in MEC of the above tissues in different tumor growth stages (simulated clinical stage). However, neither the amount of HIF-1α and vasomotor status in tissues were actually observed, nor the lymphocyte activator secreted by tumor was tested. The infiltration of CD8 + T cells and Treg+ T cells in different tissues at different stages of tumor growth have not been observed again as shown in our previous studies, specific mechanism remains to be explored and verified. Declarations Competing interests The authors declare that they have no conflict of interest. Funding This research was partially supported by grants from Tianjin Municipality Science and Technology Commission Projects (12ZCDZSY15600 to Kai Li), CSCO (Chinese Society of Clinical Oncology) Special Foundation for Tumor antiangiogenesis Therapy (Y-X2011-001 to Kai Li), National Natural Science Foundation of China (81802296 to Tingting Qin) and CSCO (Chinese Society of Clinical Oncology) Special Foundation for Tumor antiangiogenesis Therapy (Y-S2014-011 to Jing Wang). Author contributions YF performed in vivo animal experiments and wrote the manuscript; YG, YZ, SL and TQ contributed the evaluation of the data; KL and JW revised the manuscript. Availability of data and material The datasets during and/or analysed during the current study available from the corresponding author on reasonable request. References Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. 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Noman MZ, Desantis G, Janji B, Hasmim M, Karray S, Dessen P, et al. PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. The Journal of experimental medicine. 2014;211(5):781-90. Papapetropoulos A, García-Cardeña G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. The Journal of clinical investigation. 1997;100(12):3131-9. Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. The American journal of physiology. 1998;274(3):H1054-8. Namba T, Koike H, Murakami K, Aoki M, Makino H, Hashiya N, et al. Angiogenesis induced by endothelial nitric oxide synthase gene through vascular endothelial growth factor expression in a rat hindlimb ischemia model. Circulation. 2003;108(18):2250-7. Sautina L, Sautin Y, Beem E, Zhou Z, Schuler A, Brennan J, et al. Induction of nitric oxide by erythropoietin is mediated by the {beta} common receptor and requires interaction with VEGF receptor 2. Blood. 2010;115(4):896-905. Ohhashi T, Mizuno R, Ikomi F, Kawai Y. Current topics of physiology and pharmacology in the lymphatic system. Pharmacology & therapeutics. 2005;105(2):165-88. Jia Y, Qin T, Zhang X, Liu S, Liu Z, Zhang C, et al. Effect of bevacizumab on the tight junction proteins of vascular endothelial cells. American journal of translational research. 2019;11(9):5546-59. Shen G, Zheng F, Ren D, Du F, Dong Q, Wang Z, et al. Anlotinib: a novel multi-targeting tyrosine kinase inhibitor in clinical development. Journal of hematology & oncology. 2018;11(1):120. Lan W, Zhao J, Chen W, Shang H, Peng J, Lin J. Anlotinib Overcomes Multiple Drug Resistant Colorectal Cancer Cells via Inactivating PI3K/AKT Pathway. Anti-cancer agents in medicinal chemistry. 2021;21(15):1987-95. Song F, Hu B, Cheng JW, Sun YF, Zhou KQ, Wang PX, et al. Anlotinib suppresses tumor progression via blocking the VEGFR2/PI3K/AKT cascade in intrahepatic cholangiocarcinoma. Cell death & disease. 2020;11(7):573. Liang L, Hui K, Hu C, Wen Y, Yang S, Zhu P, et al. Autophagy inhibition potentiates the anti-angiogenic property of multikinase inhibitor anlotinib through JAK2/STAT3/VEGFA signaling in non-small cell lung cancer cells. Journal of experimental & clinical cancer research : CR. 2019;38(1):71. Xu P, Wang H, Pan H, Chen J, Deng C. Anlotinib combined with temozolomide suppresses glioblastoma growth via mediation of JAK2/STAT3 signaling pathway. Cancer chemotherapy and pharmacology. 2022;89(2):183-96. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3231581","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":224080520,"identity":"0aab5aed-7f64-417a-8a61-b6aefdf2d486","order_by":0,"name":"Yingfang Feng","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingfang","middleName":"","lastName":"Feng","suffix":""},{"id":224080521,"identity":"8624293f-097e-464f-9adb-b52a9f62fccf","order_by":1,"name":"Yuan Gao","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Gao","suffix":""},{"id":224080522,"identity":"523faa08-e944-4b42-9c6c-8aeb802d4d4b","order_by":2,"name":"Tingting Qin","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Qin","suffix":""},{"id":224080523,"identity":"9cd1bbaa-d685-49fa-bfe5-6ae8c2181692","order_by":3,"name":"Yan Zhang","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""},{"id":224080524,"identity":"71b4efaf-a0c0-4988-8666-58acaf78d87f","order_by":4,"name":"Shaochuan Liu","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shaochuan","middleName":"","lastName":"Liu","suffix":""},{"id":224080525,"identity":"afbf2a68-74d5-472a-8256-1fd575661807","order_by":5,"name":"Jing Wang","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":224080526,"identity":"6575e2b2-dee4-4967-91b2-75023dc1fdf5","order_by":6,"name":"Kai Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPmY2BoYEBjkGCfbGxocfiNHCBtFizCDBc7jZWIIoLUAIBEAtEultAjxEaWFnS/zwgMEgT3LmwzYGCQY7Od0Gwg47LJHAYFAsLZ3Y9qCAIdnY7ABBLewNQC1/EudJJ7YbSDAcSNxGhJbmH0BbEudJHmyT4CFOC9sxkMMSZ0swEq8lzQKkZWZPIjCQDYjwCz//MeObP4BaZhw//vDhhwo7OYJawIDxH4xlQIzyUTAKRsEoGAUEAQBPqTg+B7PHGwAAAABJRU5ErkJggg==","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-08-03 13:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3231581/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3231581/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":41371570,"identity":"c289f511-5405-42e0-898c-b952fd91e337","added_by":"auto","created_at":"2023-08-10 14:14:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7571459,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PD-L1 in MEC of ear tissue with tumor growth\u003c/p\u003e\n\u003cp\u003eImmunofluorescence of PD-L1 expression in (A) VEC and (B) LEC with tumor progression; (C) The percentage of PD-L1\u003csup\u003e+\u003c/sup\u003eVEC and (D) PD-L1\u003csup\u003e+\u003c/sup\u003eLEC in ear of the B16 tumors. CD31 labeled vascular endothelial cells and Podoplanin labeled lymphatic endothelial cells. The data were expressed as mean ± standard deviation, and the comparison between the two groups was performed by unpaired T test, *P\u0026lt;0.05, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure1.earnc.png","url":"https://assets-eu.researchsquare.com/files/rs-3231581/v1/8126efc6e3680fcd59ac6e50.png"},{"id":41371571,"identity":"11ee21ed-afca-4ce4-a059-37384e8f9068","added_by":"auto","created_at":"2023-08-10 14:14:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12710904,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PD-L1 in renal MEC with tumor growth\u003c/p\u003e\n\u003cp\u003eImmunofluorescence of PD-L1 expression in (A) VEC and (B) LEC with tumor progression; (C) The percentage of PD-L1\u003csup\u003e+\u003c/sup\u003eVEC and (D) PD-L1\u003csup\u003e+\u003c/sup\u003eLEC in renal of the B16 tumors. The data were expressed as mean ± standard deviation, and the comparison between the two groups was performed by unpaired T test, *P\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"figure2.kidneync.png","url":"https://assets-eu.researchsquare.com/files/rs-3231581/v1/d5df5506934adbd2f45e19f2.png"},{"id":41370687,"identity":"20b7ae1c-5949-4628-b535-42c0708bc7ed","added_by":"auto","created_at":"2023-08-10 14:06:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7646200,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PD-L1 of tumor MEC with tumor progression\u003c/p\u003e\n\u003cp\u003eImmunofluorescence of PD-L1 expression in (A) VEC and (B) LEC with tumor progression; The percentage of (C) PD-L1\u003csup\u003e+\u003c/sup\u003eVEC and (D) PD-L1\u003csup\u003e+\u003c/sup\u003eLEC in B16 tumor tissues. The data were expressed as mean ± standard deviation, and the comparison between the two groups was performed by unpaired T test, *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure3.tumornc.png","url":"https://assets-eu.researchsquare.com/files/rs-3231581/v1/07a819862fb3c87fbe6113d1.png"},{"id":41370685,"identity":"a8decbcb-18fc-4c76-8fd0-9637ac98a6a9","added_by":"auto","created_at":"2023-08-10 14:06:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4226720,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PD-L1 in MEC of ear tissue of B16 mice at day 22 after administration at different periods\u003c/p\u003e\n\u003cp\u003eImmunofluorescence of (A) VEC and (B) LEC express PD-L1 in ear tissues in the control group and the An5-9 group. Percentage of PD-L1 expression in (C) VEC and (D) LEC. The data were expressed as mean ± standard deviation, and the comparison between the two groups was performed by unpaired T test, **P\u0026lt;0.01, ****\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure4.earan.png","url":"https://assets-eu.researchsquare.com/files/rs-3231581/v1/0d39134267971d417cc8a617.png"},{"id":41370683,"identity":"0019aa89-05c9-481e-a418-48381445f549","added_by":"auto","created_at":"2023-08-10 14:06:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5633696,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PD-L1 in MEC of renal tissue of B16 mice at day22 in different administration groups\u003c/p\u003e\n\u003cp\u003eImmunofluorescence of (A) VEC and (B) LEC express PD-L1 in renal tissues in the control group and the An10-14 group. Percentage of PD-L1 expression in (C) VEC and (D) LEC with tumor growth. The data were expressed as mean ± standard deviation, and the comparison between the two groups was performed by unpaired T test, **P\u0026lt;0.01, ****\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure5.kidneyan.png","url":"https://assets-eu.researchsquare.com/files/rs-3231581/v1/2ed45ab1996ed4ab9d92658a.png"},{"id":41370682,"identity":"6926009d-0a7d-42b5-9513-7f13929e8b07","added_by":"auto","created_at":"2023-08-10 14:06:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5714519,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PD-L1 in MEC of tumor tissues at day22 in different administration groups\u003c/p\u003e\n\u003cp\u003eImmunofluorescence of PD-L1 expression on (A) VEC and (B) LEC of tumor tissues in the control group and the An5-9 group. Percentage of (C) VEC and (D) LEC PD-L1\u003csup\u003e+\u003c/sup\u003e in different groups. The data were expressed as mean ± standard deviation, and the comparison between the two groups was performed by unpaired T test, with **\u0026lt;0.01, ***\u0026lt;0.001, and ****\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure6.tumoran.png","url":"https://assets-eu.researchsquare.com/files/rs-3231581/v1/c93816d014e459b2dabe512f.png"},{"id":42950540,"identity":"8505af01-530b-480f-aaf7-4f3722243dbb","added_by":"auto","created_at":"2023-09-11 15:37:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5500056,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3231581/v1/fafaa12d-e50b-431d-af4d-07ad963054eb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Changes in PD-L1 expression on microvessel endothelial cells during melanoma tumor growth and the regulation by anlotinib","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePD-1 is an immunosuppressive receptor expressed on the surface of activated T cells, B cells and bone marrow cells(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e); and it\u0026rsquo;s ligand, PD-L1, is expressed on a variety of cells, including macrophages, dendritic cells, tumor cells, lymphocytes in draining lymph nodes, and non-lymphoid tissues (such as heart, lung)(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The binding of PD-L1 on tumor cell to PD-1 on T cell inhibits the activation and proliferation of T cells, leading to the depletion of CD8\u003csup\u003e+\u003c/sup\u003eT cells, inducing tumor tolerance to T cells and promoting tumor immune escape(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, the majority of research has focused on the connection between the high PD-L1 expression on tumor cells and the ideal efficacy of anti-PD-L1. However, still there are a considerable number of patients whose tumor cells with high expression of PD-L1 revealed poor therapeutic efficacy of anti-PD-L1, suggesting that there are other mechanisms affecting the prognosis beyond the PD-L1 on tumor cells(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Recent researches proved that PD-L1 on vascular endothelial cells (VEC) and lymphatic endothelial cells (LEC) could be induced by tumor and banded with PD-1 on CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes, inhibiting T cell infiltration and leading to its depletion(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), hence leading to the escape of tumor immune damage. Moreover, those PD-L1 on tumor-associated micro-vascular/lymphatic endothelial cells (MEC) could \u0026ldquo;consume\u0026rdquo; and waste the PD-L1 antibody that should be banded with the PD-L1 on tumor cells. Therefore, how to remove the inhibition on CD8\u003csup\u003e+\u003c/sup\u003e T cells by PD-L1 on the surface of tumor-associated MEC to break this \"immune barrier\" has become another key point to improve the efficacy of immunotherapy.\u003c/p\u003e \u003cp\u003eAnlotinib, a small molecule multi-targeted receptor tyrosine kinase inhibitor, which inhibited VEGFR1-3, FGFR1-4, PDGFR α and β, and C-kit, can inhibit tumor cell proliferation and anti-tumor angiogenesis(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Our previous studies have shown that anlotinib could inhibit tumor growth by down-regulating the expression of PD-L1 on tumor vascular endothelial cells to improve the infiltration and activity of lymphocytes in tumor tissues(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), which provides its potential application in synergy with immunotherapy. However, the changes of PD-L1 on VEC and LEC in tumor and normal tissues during tumor growth have not been dynamically observed, neither the effects of anlotinib on the expression of PD-L1 on MEC in tumor and normal tissues has been observed. This study aims to observe the above indexes and evaluate the optimal time of administration of anlotinib to get the best synergistic effect with immunotherapy, and potential damage by infiltrative CD8\u0026thinsp;+\u0026thinsp;cells through MEC into normal tissues, for providing the reference to clinical treatment.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCells culture and reagents\u003c/h2\u003e \u003cp\u003eB16 cells was obtained from Tianjin Medical University Cancer Institute and Hospital. Female C57BL/6J mice aged 6\u0026ndash;8 weeks were purchased from the SPF (Beijing) Biotechnology Co.,Ltd. All the experimental program conforms to the tumor hospital of tianjin medical university laboratory animal ethics rules. Anlotinib was provided as gift by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. The cells were cultured with DMEM medium containing 10% fetal bovine serum (FBS) at 37\u0026deg;C in a humidified incubator containing 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo experiment\u003c/h2\u003e \u003cp\u003eTo construct a tumor-bearing mouse model of B16 cells, we injected 1\u0026times; 10\u003csup\u003e6\u003c/sup\u003e/100 \u0026micro; L B16 cells subcutaneously into the right groin region of each mouse. Tumor growth was observed every day for several weeks. Based on the previous studies, tumor volume was calculated by the formula of volume\u0026thinsp;=\u0026thinsp;length \u0026times; width \u0026times; width /2(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), and the 22nd day was set as the end time of mice\u0026rsquo;s survival. The tumor growth curve was drawn, and B16 melanoma was divided into three growth stages according to the tumor volume growth rate(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), approximately simulating the clinical T stage, namely day5-9, day 10\u0026ndash;14, and day16-20. In order to explore when the administration of anlotinib has an optimal effect, we established B16 xenograft tumor model and divided mice into four groups, NC (PBS, 1.5 mg/kg, once daily), An5-9 (anlotinib, 1.5 mg/kg, once daily for day5-9), An10-14 (anlotinib, 1.5 mg/kg, once daily for day10-14) and An16-20 (anlotinib, 1.5 mg/kg, day16-20 once daily), respectively. We compared the changes of MEC PD-L1 in normal mice and non-drug-treated tumor-bearing mice at the 10th, 15th and 22nd days as observation points. For mice in each treatment group, the 22nd day after tumor bearing was selected as the observation point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSelection of tissue (organs) to observe the PD-L1 on MEC\u003c/h2\u003e \u003cp\u003eAccording to the previous research, characteristics of PD-L1 expressed by VEC and LEC are different, which suggested that tumor might have different effects on them, so VEC and LEC were selected respectively to be observed. Tumor and normal tissue can both be influenced by medicine, so we choose both of them. In the common view of the organs with side effects of immunotherapy, we choose ear tissue representing the peripheral skin and kidney tissue representing the viscera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining (IF)\u003c/h2\u003e \u003cp\u003eThe fresh frozen sections were rewarmed at room temperature for 10 minutes, then the tissues were fixed with 4% paraformaldehyde precooled 4℃ for 15 minutes. The frozen tissue samples after fixation were permeated with 0.2% TritonX-100 diluted with PBS for 10 minutes. Washing with PBS 3 times, for 5 minutes each time, and all sections were incubated with PBS block solution containing 1% BSA, 0.01% Triton X-100 and 10% FBS at room temperature for 1h. The slices were incubated with primary antibody overnight at 4℃. The primary antibodies used were as follows: Rat anti-mouse CD31(AB56299, Abcam), Syrian hamster anti-mouse Podoplanin (AB11936, Abcam), rabbit anti-mouse PD-L1 (LS-C746930, LifeSpan). The sections were then rewarmed at room temperature for 30 min and washed in PBS 3 times, 5 min each. The tissue sections were stained with fluorescent secondary antibody and incubated at room temperature for 1h. The following secondary antibodies were used: donkey anti-rat AF488 (A21208, Invitrogen), donkey anti-Syrian hamster AF546 (A21111, Invitrogen), donkey anti-rabbit AF647 (A31573, Invitrogen); then washed in PBS for 3 times. Finally, all the sections were stained with anti-fluorescence quencher (including DAPI). The stained sections were stored at -20℃, protected from light, and Zeiss Imaginer-Z2 was used for image acquisition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation the percentage of PD-L1\u0026thinsp;+\u0026thinsp;MEC in tissues\u003c/h2\u003e \u003cp\u003eCD31(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and Podoplanin(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)was used to label vascular endothelial cells and lymphatic endothelial cells, respectively. To assess the percentage of CD31\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e and Podoplanin\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e in tissues, the sites with the most abundant blood vessels and lymphatic vessels were selected under 10-fold microscope, 3\u0026ndash;5 independent fields were randomly selected under 40-fold objective lens, and the percentage of CD31\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells in all CD31\u003csup\u003e+\u003c/sup\u003e cells and Podoplanin\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells in all Podoplanin\u003csup\u003e+\u003c/sup\u003e cells was calculated. Take the average and use it as the final result for each section(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were calculated using SPSS v.24 (IBM Corp), statistical graphs and analyses were generated using GraphPad Prism 8 (USA, GraphPad Software). All measurement data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, unpaired T test was used for comparison between two groups of data, and one-way ANOVA was used for comparison between multiple groups of data. Wilcoxon rank sum test was used for non-normally distributed data. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression of PD-L1 on VEC and LEC of normal tissues in naive mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining showed that VEC PD-L1, namely CD31\u003csup\u003e+\u003c/sup\u003e PD-L1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e(%) expression was 68.41\u0026plusmn;18.44 in the ear tissues of non-tumor burden mice. The expression of LEC-PD-L1, namely Podoplanin\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/ Podoplanin\u003csup\u003e+\u003c/sup\u003e(%), was 33.92\u0026plusmn;20.17 (Fig.1). The positive expression rate of VEC-PD-L1 and LEC-PD-L1 in kidney tissues of non-tumor burden mice was 14.25\u0026plusmn;5.864 and 24.16\u0026plusmn;17.75 (Fig.2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePD-L1 expression of VEC and LEC in ear tissues of tumor-bearing mice during tumor growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD31\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/CD31+(%) in PBS group were 16.10\u0026plusmn;5.933, 16.13\u0026plusmn;9.228 and 12.63\u0026plusmn;5.444 at day 10, 15 and 22, respectively. Compared with normal mice, tumor-bearing mice VEC PD-L1 expression was significantly decreased (P\u0026lt;0.0001) (Fig.1A, C). Podoplanin \u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/ Podoplanin \u003csup\u003e+\u003c/sup\u003e(%) in PBS group on day 10, 15 and 22 were 17.09\u0026plusmn;3.657, 18.04\u0026plusmn;12.44 and 17.75\u0026plusmn;11.39, respectively. The expression of LEC-PD-L1 declined on day 10 and 22 compared with ear of normal mice (P=0.0248, P=0.0295, respectively) and there was no significant difference in the expression of LEC-PD-L1 on day 15 in tumor-bearing mice compared with normal mice (P\u0026gt;0.05) (Fig.1B, D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePD-L1 expression of VEC and LEC in renal tissues of B16 mice during tumor growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD31\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e(%) in PBS group were 31.52\u0026plusmn;12.52, 27.91\u0026plusmn;14.45 and 14.48\u0026plusmn;3.930 on day 10, 15 and 22, respectively. Compared with vec-PD-L1 in renal tissues of normal mice on day 0, there was no significant difference in VEC-PD-L1 expression after tumor bearing (P\u0026gt;0.05) (Fig.2A, C). PODOPLANIN\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/PODOPLANIN\u003csup\u003e+\u003c/sup\u003e(%) in PBS group were 23.28\u0026plusmn;8.071, 25.70\u0026plusmn;14.15 and 35.87\u0026plusmn;14.63 at day 10, 15 and 22, respectively. Compared with day 0 (normal mice), LEC-PD-L1 in renal tissue tended to be up-regulated after tumor loading, with a near significant difference at day 22 (P=0.2408)\u0026nbsp;(Fig. 2B, D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVEC and LEC expressed PD-L1 in B16 tumors with tumor growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to previous literature, both VEC and LEC of tumor tissues express PD-L1, which is induced by IFN-\u0026gamma; that produced by tissue-infiltrating, antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells(14). However, little research on the dynamic changes of MEC PD-L1 with tumor growth, so the expression of CD31\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e(%) and PODOPLANIN\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/ PODOPLANIN\u003csup\u003e+\u003c/sup\u003e(%) were compared at days 10, 15 and 22 after tumor cells were inoculated. We found that with tumor progression, VEC PD-L1 was escalate. Compared with the quantitative value on day 10, VEC PD-L1 on day15 was not significantly up-regulated (P=0.3899), while that on day22 was significantly up-regulated (***P= 0.0005) (Fig. 3A, C). LEC PD-L1 was also increased in tumor tissues, and compared with the percentage of day10, it was not significantly up-regulated at day15 (P=0.9944), while significantly up-regulated at day22 (**P=0.0024). Tumor-associated MEC PD-L1 were increased across the progression of tumor (Fig. 3B, D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of anlotinib given at different stages of tumor on PD-L1 expression of VEC and LEC in ear tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with the control group, the proportion of CD31\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e(%) was slightly up-regulated in all treatment groups, without statistical significance (P\u0026gt;0.05) (Fig. 4A, C). In Comparison with the control group, the expression of Podoplanin \u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/ Podoplanin\u003csup\u003e\u0026nbsp;+\u003c/sup\u003e(%) was significantly up-regulated in the early treatment group (**P=0.0019) (Fig. 4B, D). It was slightly up-regulated in the middle dosing group, and slightly down-regulated in the late dosing group, with no statistical significance (P\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of anlotinib given at different stages of tumor on PD-L1 expression of VEC and LEC in renal tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn comparison with the control, the rate of CD31\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e(%) was slightly upregulated in the treatment groups, without statistical significance (P\u0026gt;0.05) (Fig.5A, C). While Podoplanin\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e/ Podoplanin\u003csup\u003e+\u003c/sup\u003e(%) was down-regulated in all treatment groups compared with the control group, (Fig.5B, D), and there were statistically significant differences in the early and middle dose-adding groups (P=0.0232, P=0.0037). There was no statistical significance in late treatment group (P\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of anlotinib given at different stages of tumor on PD-L1 expression of VEC and LEC in tumor tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous study found that anlotinib could attenuate the expression of PD-L1 in microvascular endothelial cells. Compared with the day22 in control group, VEC-PD-L1 was down-regulated in each period after administration, and the differences were statistically significant (****P\u0026lt;0.0001, ***P=0.0002, **P=0.0097). And the most obvious down-regulation was observed at early treatment group (Fig.6A, C). In comparison with the control group, LEC-PD-L1 was down-regulated in each period in treatment group with the significant differences (****P\u0026lt;0.0001, ****P\u0026lt;0.0001, ****P\u0026lt;0.0001). There was no statistically difference between treatment groups at each stage (Fig.6B, D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eRegulation of PD-L1 in endothelial cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe application of immune checkpoint inhibitors (ICIs) have promoted the treatment of cancer. ICIs targeting programmed death ligand 1 (PD-L1) have shown striking anti-tumor efficacy in a variety of cancers(17). While only a part of patients benefit from it in clinical implementation(18-21).\u0026nbsp;This may be related to the fact that MEC can also express PD-L1 and form an \u0026quot;immune barrier\u0026quot; to prevent active lymphocytes from infiltrating into tumors. Conversely, down-regulation of PD-L1 expression on MEC could weaken this barrier and promote CD8\u003csup\u003e+\u003c/sup\u003eTeff cells to infiltrate the tumor and improve the immunotherapy effect(10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn MECs, recent study(14)\u0026nbsp;has found that LEC and VEC can express PD-L1 in melanoma and inflammatory skin, VEC can express PD-L1 systemically in all conditions, regardless of whether or not the tumor is carried and what the type of tumor,\u0026nbsp;while LEC demonstrated high specificity to the local microenvironments, even high expression of LEC-PD-L1 in normal tissues only on the ipsilateral side of the lesion (\u0026quot; near area \u0026quot;), but low expression of LEC-PD-L1 in normal tissues on the contralateral side of the lesion (\u0026quot; far area \u0026quot;) were found(14).\u0026nbsp;The authors believe that it was due to the different properties of the two types of MEC. LEC is more likely to show an \u0026quot;active response\u0026quot; to surrounding lesions than the \u0026quot;passive\u0026quot; of VEC. In addition, the supply of blood and oxygen in different tissues are also important factors that affect the expression of PD-L1 in MEC. The expression of PD-L1 is regulated by a variety of mechanisms, the following are elaborated separately.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(1) CD8\u003csup\u003e+\u003c/sup\u003eT cells and secreted factors in local tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accumulation of antigen-specific T cells and the activation of local TCR increased the concentration of IFN-\u0026gamma; in the infiltrated tissues, and IFN-\u0026gamma; activated LEC by inducing PD-L1 expression through the JAK/STAT pathway; subsequently, LEC-PD-L1 limited the infiltration of activated CD8\u003csup\u003e+\u003c/sup\u003eT cell and protected its \u0026quot;targeted tissue\u0026quot; in turn. Several studies have confirmed that endogenous expression of interferon regulatory factor (IRF-1) is necessary for constitutive and inducible B7-H1 transcription and regulates IFN-\u0026gamma; induced B7-H1 (CD274, PD-L1) through the JAK/STAT pathway(22, 23).\u0026nbsp;Anti-angiogenic drugs could facilitate the accumulation of perivascular activated CD8\u003csup\u003e+\u003c/sup\u003eT cells to up-regulate the expression of endothelial PD-L1 through IFN-\u0026gamma;(24). It should be noted that such a regulatory effect occurs and exists only between T cells and their adjacent LEC, and their spatial distribution must be very coincidence. However, the regulatory effect of T cells on distant LEC is not strong as well as weaker on VEC(14). Therefore, this effect mainly regulates the local and peripheral tissues near the lesion(14).\u0026nbsp;In this study, we observed that consistent with the trend in tumors, the LEC PD-L1 of kidney in B16 melanoma model was increased at day 22 of tumor growth, closing to a statistically significance. So, we speculated that this pathway mainly regulates the expression of PD-L1 in LEC of tumor, and kidney tissue that easier obtain the regulators form tumor through the rich of blood perfusion (equivalent to \u0026quot;near tumor tissues\u0026quot;) so that more CD8\u003csup\u003e+\u003c/sup\u003eT cells could infiltrate into tissue to secrete IFN-\u0026gamma;. However, LEC PD-L1 may not be mainly regulated by this mechanism in ear tissues (equivalent to \u0026quot;distal tumor tissues\u0026quot;) that are not rich in blood flow and receive too little chemokines (such as CCL4, CCL5 and CXCL9(25, 26)\u0026nbsp;to recruit CD8\u003csup\u003e+\u003c/sup\u003eT cells, neither may be regulated by such way in VEC with \u0026ldquo;passive response\u0026rdquo; to local immune environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(2) HIF-1 \u0026alpha; and VEGF-NO pathway\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter post-translational modification of HIF-1\u0026alpha; (hypoxia-inducible factor 1\u0026alpha; subunit) by prolyl hydroxylase under normoxic conditions, the hydroxylase binds to pVHL (von Hippel-Lindau protein) to induce ubiquitination of HIF-1\u0026alpha;(27).\u0026nbsp;Hypoxia is common in tumors and some relatively ischemic tissue (organ), in this way, the binding of pVHL to HIF-1\u0026alpha; and ubiquitin degradation is attenuated, which results in the accumulation of HIF-1\u0026alpha;. Simultaneously, it forms a dimer with HIF-1\u0026beta;, which is subsequently transferred to the nucleus(28), where HIF-1\u0026alpha; binds to a transcriptionally active hypoxia response element (HRE) in the proximal promoter of PD-L1, leading to activated transcription of PD-L1(29).\u0026nbsp;VEGF produced by tumor tissues can upregulate eNOS (endothelial nitric oxide synthase) in endothelial cells to elicit the release of NO by endothelial cells and loosen smooth muscle, resulting in microvasodilation and increased vascular perfusion(30-34), relieving hypoxia in near and distant tissues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(3) PI3K-AKT pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivated PI3K/AKT pathway can up-regulate PD-L1 expression on vascular endothelial cells\u0026nbsp;(10, 35).\u0026nbsp;This pathway mainly plays a role in local tumor tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegulation of dominance in different tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of VEC PD-L1 in tumor tissues increased with tumor growth, the regulatory mechanism could be the following: (1) local lesions oxygenation decreased,\u0026nbsp;the hypoxia-HIF-1\u0026alpha;-PD-L1 pathway was active; (2) the secretion of VEGF increased in tumor tissues, which could activate\u0026nbsp;VEGF-PI3K/ AKT-PD-L1\u0026nbsp;pathway; (3) the IFN-\u0026gamma;-JAK /STST-PD-L1 pathway, which was related with CD8\u003csup\u003e+\u003c/sup\u003eT cells. Similar to VEC, LEC-PD-L1 in tumor tissues was also enhanced with the mechanism described above.\u003c/p\u003e\n\u003cp\u003eThe expression of PD-L1 in MEC of ear tissue in tumor-bearing mice was significantly down-regulated compared with normal mice, especially in VEC. This can be attributed to the fact that ear tissue is a terminal circulating tissue and in a state of relative hypoxia. Thus, the regulatory pathway of HIF-1\u0026alpha; played a dominant role in regulation, with high expression of PD-L1 in MEC. However, when amounts of VEGF was secreted by carrying tumors, for the ear tissue of poor terminal microcirculation, their relatively contracted micrangium may be expanded by VEGF-eNOS-NO regulation more than the vessels in blood-rich, oxygenated renal tissue. So, in the case of ear, the control effect of vascular vessel is also stronger than lymph vessels; the expression of VEC PD-L1 was decreased in ear but not in renal tissue in tumor-burdened mice.\u003c/p\u003e\n\u003cp\u003eVEC PD-L1 expressed without significant change in renal tissues between normal and tumor-bearing mice. As in normal mice, renal tissue has adequate blood perfusion and oxygenation with low HIF-1\u0026alpha;; while in tumor-bearing mice, oxygen supply could be increased only slightly, vasodilatation by VEGF-eNOS-NO pathway was limited, so HIF-1\u0026alpha; was not significantly decreased. Meanwhile, the expression of LEC PD-L1 was up-regulated in renal tissues (especially at the 22d of advanced tumor), the reason could be numerous lymphocytes activating factors produced by tumors at this time to attract lymphocytes into renal tissues (as a blood-rich \u0026ldquo;near tumor tissue\u0026rdquo;) and secrete IFN-\u0026gamma; to activate the JAK/STAT pathway in renal LEC to up-regulate the expression of PD-L1. Meanwhile, NO had a better impact on vascular dilation than on lymph vessel. The IFN-\u0026gamma;-JAK /STST-PD-L1 pathway activated by CD8\u003csup\u003e+\u003c/sup\u003eT cells played the major role, promoting the upregulation of LEC-PD-L1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegulation of anlotinib in different tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnlotinib targets VEGFR2, PDGFR\u0026beta; and FGFR1(36), and our previous studies have demonstrated that anlotinib can down-regulate PD-L1 expression in VEC through inactivation of Akt(10). Other studies have also confirmed that anlotinib can inhibit the activation of PI3K/Akt pathway(37, 38).\u0026nbsp;In addition, several studies have found that anlotinib can inhibit JAK2/STAT3/VEGFA signaling pathway(39, 40). However, the distribution of anlotinib is significantly affected by the status of blood perfusion. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression of VEC-PD-L1 was slightly upregulated in ear tissues of each treatment group in tumor-bearing mice. This is probably because, on the one hand, anlotinib targets VEGFR so the VEGF-eNOS-NO pathway was limited, ear vessels return to the state of relative hypoxia; on the other hand, as a small-molecule drug, a little anlotinib may reach to VEC and reduce the expression of VEC-PD-L1 by inhibiting the PI3K/AKT pathway.\u0026nbsp;The ear LEC-PD-L1 was significantly upregulated in the early treatment group, while was slightly down-regulated in the late treatment group, which may be due to the inhibition of VEGF-NO pathway in early stage of tumor to form more obvious impact as aforementioned on more ischemic LECs. When the tumor grew to advanced stage, anlotinib could not completely inhibit the impact of VEGF, the ear lymphatic vessels were relatively dilated, and HIF-1\u0026alpha; was degraded. Therefore, the expression of LEC-PD-L1 in the advanced treatment group was slightly lower than that in the control group.\u003c/p\u003e\n\u003cp\u003eThe expression of VEC-PD-L1 in renal tissues was slightly upregulated in each treatment group. We speculated that anlotinib inhibited the expression of VEC-PD-L1 by inhibiting PI3K/AKT pathway, but inhibited VEGFR to weaken the VEGF-eNOS-NO pathway, so activated HIF-1\u0026alpha;-PD-L1 pathway. The two effects interact with each other so that VEC-PD-L1 kept approximately stable.\u0026nbsp;The renal LEC-PD-L1 was down-regulated in treatment groups, which might be attributed to the fact that anlotinib could act on the renal lymphatic vessels and inhibit the activated PI3K/AKT and IFN-\u0026gamma;-JAK/STAT pathway. Although anlotinib also inhibits lymphatic vessel expansion by inhibiting VEGFR, leading to hypoxia and upregulating HIF-1\u0026alpha;, it was obviously weaker than the same impact on VEC.\u003c/p\u003e\n\u003cp\u003eIn tumor tissues, the VEC-PD-L1 declined in anlotinib groups. Based on the results of previous studies, we analyzed that anlotinib inhibited PI3K/AKT pathway and improved hypoxia to decrease HIF-1\u0026alpha; by inhibiting VEGF. LEC-PD-L1 in tumor tissue was also down-regulated for the similar mechanisms and the inhibition of JAK/STAT signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEarly treatment of anlotinib may enhance the therapeutic efficacy of PD-L1 antibody\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn present study, the expression of PD-L1 on tumor tissues VEC and LEC was upregulated with tumor progression, so both of the expression of PD-L1 on VEC and LEC can constitute an \u0026quot;immunosuppressive barrier\u0026quot; of tumor like our previous results (10). Anlotinib can significantly down-regulate PD-L1 of MEC when administered at the early stage of tumor growth, which may help to remove the immune barrier formed by microvessels expressed PD-L1 in tumor microenvironment, promote more Teff cells to infiltrate tumor tissues out of blood and lymphatic vessels.In this way, its combination with anti-PD-1 / anti-PD-L1 drugs can achieve optimal therapeutic effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePotential damage of anlotinib on different organs in immunotherapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous study showed that the down-regulation of PD-L1 expression on VEC by anlotinib can accelerate the infiltration of CD8cells in tissues and contribute to tumor control. However, the more immune cells infiltrate in normal tissue, the more attacks could happen. According to the dynamic change of PD-L1 expression in MEC of ear and kidney tissues in different groups, compared with control group, LEC-PD-L1 in ear was significantly up-regulated in the early administration group, while there was no significant difference in middle and late administration group.\u0026nbsp;Therefore, we speculated that anlotinib administration at different stages may not cause severe immune \u0026quot;toxification\u0026quot; in peripheral tissues (distal tumor tissues, such as ear skin). However, LEC-PD-L1 in kidney was down-regulated. Therefore, it is necessary to be vigilant that drug administration may cause immunotoxicity to renal tissues to some degree.\u003c/p\u003e\n\u003cp\u003eIn summary, in present study, we elucidated the changing trend in PD-L1 expression on MECs in tumor and normal tissue, and the effect of anlotinib on the various tissue (organ)s during the tumor growth. We believe our discovery should be valuable and referential for determining the optimal administrative time of \u0026ldquo;immuno-efficacy-accelerating agent\u0026rdquo; of anlotinib to cut down the \u0026ldquo;immune barrier\u0026rdquo; of PD-L1 on MEC (especial on LEC) to reinforce the PD-L1 antibody, and avoiding the toxicity of PD-L1 antibody in normal tissue.\u003c/p\u003e"},{"header":"Limitation of this study","content":"\u003cp\u003eThis study only observed the expression of PD-L1 in MEC of the above tissues in different tumor growth stages (simulated clinical stage). However, neither the amount of HIF-1\u0026alpha; and vasomotor status in tissues were actually observed, nor the lymphocyte activator secreted by tumor was tested. The infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells and Treg+ T cells in different tissues at different stages of tumor growth have not been observed again as shown in our previous studies, specific mechanism remains to be explored and verified.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by grants from Tianjin Municipality Science\u003c/p\u003e\n\u003cp\u003eand Technology Commission Projects (12ZCDZSY15600 to Kai Li), CSCO\u003c/p\u003e\n\u003cp\u003e(Chinese Society of Clinical Oncology) Special Foundation for Tumor\u003c/p\u003e\n\u003cp\u003eantiangiogenesis Therapy (Y-X2011-001 to Kai Li), National Natural Science\u003c/p\u003e\n\u003cp\u003eFoundation of China (81802296 to Tingting Qin) and CSCO (Chinese Society of Clinical Oncology) Special Foundation for Tumor antiangiogenesis Therapy (Y-S2014-011 to Jing Wang).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYF performed in vivo animal experiments and wrote the manuscript; YG, YZ, SL and TQ contributed the evaluation of the data; KL and JW revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets during and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFreeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. 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Cancer chemotherapy and pharmacology. 2022;89(2):183-96.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"vascular endothelial cells (VEC), lymphatic endothelial cells (LEC), PD-L1 melanoma Anlotinib","lastPublishedDoi":"10.21203/rs.3.rs-3231581/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3231581/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo evaluate the expression of PD-L1 on MEC in tumor and normal tissues, IF was used to detect the expression of PD-L1\u003csup\u003e+\u003c/sup\u003eCD31\u003csup\u003e+\u003c/sup\u003e VECs and PD-L1\u003csup\u003e+\u003c/sup\u003e Podoplanin \u003csup\u003e+\u003c/sup\u003e LECs in tumor, ear and kidney tissues. We found that the MEC PD-L1 in tumor showed an upward trend with tumor progression. The expression of PD-L1 in MEC in normal tissues is associated with the type of microvessels and tumor progression. The MEC PD-L1 expression in tumor is significantly down-regulated at 22th day in An5-9 group. In the early treatment group (An5-9), anlotinib increased the LEC PD-L1 but had no significant effect on VEC PD-L1 of ear, while decreased the LEC PD-L1 in kidney. In conclusion, the MEC-PD-L1 in melanoma increased with tumor growth and was downregulated significantly by early treatment of anlotinib, which proposes a potentiality of its enhancement on the therapeutic efficacy of anti-PD-L1 antibody. Anlotinib had no significant effect on the MEC PD-L1 of ear excepting the upregulation of LEC PD-L1 in early treatment group, while decreased such expression in kidney, hence the risk of possible renal damage by CD8 + T cells in the treatment of anlotinib should be bewared.\u003c/p\u003e","manuscriptTitle":"Changes in PD-L1 expression on microvessel endothelial cells during melanoma tumor growth and the regulation by anlotinib","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-10 14:06:03","doi":"10.21203/rs.3.rs-3231581/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a513733a-c235-43c4-aff4-bd98f5e79054","owner":[],"postedDate":"August 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-11T15:29:32+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-10 14:06:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3231581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3231581","identity":"rs-3231581","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}