The Study On 2-hexyl-4-pentylenic Acid (HPTA) Stimulates The Radiotherapy-induced Abscopal Effect By Reprogramming Tumor-associated Macrophages Polarization

ObjectivesAccording to the latest statistics of the global cancer burden released by the World Health Organization in 2020,the number of new cases of breast cancer has increased rapidly and became the largest cancer in the world.Radiotherapy(RT)is a widely used and highly effective treatment for cancer,which main mechanism is to cause the death of tumor cells directly by damage to DNA.However,in recent years,radiation-induced abscopal effect has attracted the attention of clinicians and oncologists.Abscopal effect refers to the spontaneous regression of tumors in the unirradiated region after local radiotherapy to the primary tumor site,the mechanism is thought to be a systemic anti-tumor immune response stimulated by RT.However,in fact,because radiotherapy can also induce immunosuppression,the occurrence of distal effect is relatively rare.Therefore,how to effectively overcome radiation-induced immunosuppression,enhance the systemic antitumor efficacy of local RT and promote the occurrence of distal effects is the focus in this field.Tumor-associated macrophages(TAMs)are the main cell group in the Tumor microenvironment,accounting for about 50%of the Tumor stroma.Most of the TAMs in tumors are M2 type,which is closely related to the growth,metastasis and invasion of tumors.But TAMs are a kind of highly plastic immune cell population,which can switch between anti-tumor M1 type and pro-tumor M2 type.M1 type macrophages can induce the release of pro-inflammatory cytokines and play a role in killing tumor cells.It is speculated that remodeling the polarization of TAMs to anti-tumor M1 phenotype may promote the occurrence of abscopal effect.Thus,it is urgent to find an agent that can specifically promote the differentiation of TAMs into anti-tumor M1 type and initiate the abscopal effect.Our previous research has shown that 2-hexyl-4-pentylenic acid(HPTA),a valproic acid derivative,possesses histone deacetylase inhibitor(HDACi)functions,is a novel pharrmacotherapeutic for breast cancer cells by decreasing the activity of DNA repair proteins and increase apoptotic cell death[26].A recent study found that TMP195,a class Ⅱa HDACi,inhibited breast tumor cell proliferation through regulating the polarization of TAMs to an anti-tumor M1 phenotype,and induced normalization of blood vessels in breast tumors to reduce metastasis[27],thus suggesting that HDACis may be able to regulate the polarization of TAMs,but it has not been reported whether HPTA has the ability to influence the differentiation of TAMs,and whether RT combined with HPTA can stimulate the occurrence of radiation-induced abscopal effects.The purpose of this project is to study whether HPTA combined with RT can stimulate the occurrence of abscopal effect,and on this basis to explore its mechanism,so as to provide reliable theoretical guidance for enhancing the systemic anti-tumor effect of local radiotherapy,which has important clinical significance for the prevention and treatment of breast cancer patients with tumor metastasis.Methods1.The establishment of radiation-induced abscopal effect in animal50-day-old female SD rats(body weight 150±15g)were given 20mg dimethylbenzoanthracene(DMBA),an environmental carcinogen dissolved in vegetable oil,by oral gavage.Primary breast tumors of the rats were successfully induced about 90 days after DMBA gavage.Subsequently,radiation-induced abscopal effect animal model was established on the basis of primary breast tumor model.Twenty rats with two or more tumors were randomly assigned into four groups:control group,HPTA group alone,RT group alone,and RT+HPTA group.Control group,which means without any treatment.The HPTA group and RT+HPTA group were intraperitoneally injected 20mg/kg HPTA at the same time for 6 consecutive days,for a total of 12 times with an interval of 12 hours each time.For IR,only one tumor was selected as the irradiated tumor,and local precise irradiation was performed.The remaining tumors were unirradiated tumors.Respectively,the total radiation dose of RT group and combined group was 8Gy,and the treatment method was dose fractionation(2Gy/time/day,4 times in total).Rats were irradiated at the same time on the second day after administration.The rats were sacrificed on day 10 and the rest were monitored weekly for the responses of tumors were until day 35.2.Tumor volume measurementTumor change was recorded weekly after irradiation and drug treatment,and irradiated and unirradiated tumor volume was calculated according to the clinical standard formula:tumor volume(mm3)=Length(L)×Width(W)2/2.3.Histopathology observation of tumor tissue in unirradiated areasMorphological changes of breast tumor were observed by HE staining.4.Detection of proliferative ability of tumor cells in unirradiated areas5-Bromo-2-deoxyuridine(BrdU)was injected intraperitoneally at a dose of 100 mg/kg 24 hours before the rats were killed,and the expression of BrdU and Ki67 proliferation markers in the distal non-irradiated tumor tissue was detected by immunohistochemical staining.5.Phenotype identification of macrophages in unirradiated tumors①Identification of macrophages:The macrophage markers F4/80,CD68 and CD11b were detected by immunohistochemistry and immunofluorescence co-staining,and the number and distribution of macrophages were studied.②Identification of macrophage polarization status:The expression of surface markers(CD86/CD163+)and functional markers(iNOS/Arg-1+)of M1/M2 macrophages were detected by immunofluorescence co-staining,qRT-PCR and Western blotting,respectively,to study the polarization of macrophages.6.Detection of inflammatory factors associated with tumor-associated macrophages in unirradiated tumorsThe expression levels of inflammatory cytokines,such as TNF-α,IFN-γ,IL-12,IL-6,IL-10 and TGF-β,which are related to the activity of Ml and M2 macrophages were detected by qRT-PCR.7.Detection of tumor angiogenesis in unirradiated areasThe expression level of IFN-γ protein with anti-angiogenic properties in tumor tissues was detected by western blotting.Vascular markers CD31 and CD34 were used to evaluate vascular density and integrity by immunofluorescence staining.The protein expression level of IFN-γ in tumor tissues was detected by Western blot assay.Surface markers of vascular endothelial cells CD31 and CD34 were used to assess vascular density and integrity by immunofluorescence staining.Statistical analysisData were expressed as mean±standard deviation(SD)The difference between groups was determined by One-way analysis of variance(ANOVA).P<0.05 or P<0.01 indicated a statistically significant difference.Results1.HPTA can stimulate RT-induced abscopal effect and significantly delay the growth of unirradiated breast tumorThe morphological structure of the DMBA-induced breast tumor tissue was observed by HE staining,in contrast to normal breast tissue with a small number of mammary ducts and complete acinus,we found a lot of abnormal hyperplasia of breast ducts and interstitial fibrosis in the tumor tissue.The acinar structure of the breast tissue disappeared,and the cells in the breast ducts proliferated in large numbers and were arranged disorderly,showing the typical tumor tissue cell morphology,which indicated that the mammary carcinoma in rats was successfully induced by DMBA.Subsequently,the abscopal effect model induced by radiotherapy for breast cancer in rats was established(see Method 1).By observing changes in tumor volume over 35 days,we found that RT caused significant growth delay in the irradiated tumor(P<0.01)but had no effect on the unirradiated tumors.RT combined with HPTA showed a significant effect on the irradiated tumor growth,resulting in tumor inhibition in comparison with RT alone(P<0.0001).Importantly,the growth of unirradiated tumors in the RT+HPTA combination group was also significantly inhibited as compared with the corresponding RT-only group(P<0.01).The results indicated that HPTA can trigger RT-induced an abscopal effect.2.HPTA combined with RT can effectively inhibit the proliferation of tumor cells and promote the infiltration of myeloid-derived TAMs in distal unirradiated tumorsBy HE staining,in the control group,we observed abnormal hyperplasia with a large number of malignant cells and interstitial fibrosis.There were only a few cavities in the tumor tissue in the group treated with RT only,and HPTA administration alone caused a small amount of cavity necrosis,which was no significant difference compared with the control group,whereas it was found that a mass of vacuoles necrosis in RT+HPTA group,indicating that combined treatment of HPTA and RT can result in significant necrosis of tumor tissue.Immunohistochemical staining of two proliferation markers,BrdU and ki67,showed that a large number of proliferating cells in distant unirradiated tumor tissue appeared in the DMBA group,and the number of proliferating cells in the HPTA-only group and RT-only group was decreased(P<0.05),but there was no significant difference,while HPTA when combined with RT resulted in a dramatic decrease in proliferating cells in unirradiated tumor tissues(P<0.01).The results showed that the ability of cell proliferation in tumor was significantly reduced after RT combined with HPTA.Immunohistochemical staining of specific macrophage markers,F4/80 and CD68,showed that in the control group,there were few macrophages in tumor tissue and a slight increase in macrophages in the tumor tissues of the HPTA-only and RT-only group(P<0.05),however,HPTA when combined with RT resulted in a dramatic increase in the macrophages(P<0.01)and they were mainly located in the vacuole regions formed by tumor necrosis,indicating that the combined treatment may effectively promote the recruitment and infiltration of the macrophages to tumor stroma for eliminating of tumor cells.Immunohistochemistry and immunofluorescence co-staining of myeloid cell marker CD11b and macrophage marker F4/80 showed that RT combined with HPTA caused a significant increase in the number of CD11b+monocytes compared with the other groups(P<0.01),and the amount of cell with both CDllb and F4/80 staining in the RT+HPTA group was significantly higher than that of HPTA-only,RT-only and control group(P<0.01).In summary,our results showed that RT+HPTA can induce an increase in the population of infiltrated myeloid-derived TAMs into tumor microenvironment,suggesting that infiltrated TAMs in unirradiated tumor maybe responsible for the abscopal effect.3.HPTA can reprogram TAMs in the unirradiated region to polarize to anti-tumor M1 phenotype and reverse the decrease of M1/M2 macrophages ratio induced by RT.The results of qRT-PCR showed that compared with the control group,CD86(M1 phenotype)expression was increased and CD 163(M2 phenotype)expression was decreased in the HPTA group,while Ml was decreased and M2 was increased in RT group alone.The expression of Ml in HPTA+RT group was significantly higher than that in RT alone group(P<0.01),and M2 decreased significantly,and the ratio of M1/M2 increased.qRT-PCR and Western blot showed that the ratio of iNOS/Arg-1 of M1 and M2 functional markers also showed the same trend(P<0.01).The results above indicated that the application of HPTA could reprogramme the differentiation of TAMs within the tumor microenvironment,skewing the TAMs differentiated to M1 subtype.RT-only could promote TAMs differentiated into pro-tumor M2 type.The combined application of RT and HPTA could inhibit the differentiation of TAMs to M2 type compared with RT-only.We next quantified the cytokines associated with TAMs activity in the unirradiated tumor tissue using qRT-PCR analysis.The results showed that compared with the RT-only group,the cytokines related to M2 macrophages immune activity,TGF-β(P<0.01)and IL-10(P<0.01),were significantly suppressed when HPTA was combined with RT,while the pro-inflammatory cytokines IFN-y,TNF-α and IL-12 related to M1-type macrophages were significantly increased.The results of immunofluorescence co-staining of F4/80 and CD86 markers also further confirmed that the combination of HPTA and RT could TAMs differentiation into the anti-tumor M1 phenotype and reverse the decrease of M1/M2 macrophages ratio induced by RT,thereby enhancing the anti-tumor immune effect.4.HPTA can inhibit neovascularization in unirradiated tumors.The result of qRT-PCR and western blot showed that the mRNA and protein levels of IFN-γ with anti-angiogenic properties in combination group were significantly higher than those in other groups(P<0.01).By immunofluorescence staining of CD31 and CD34,the surface markers of neovascular endothelial cells,we found that size and density of CD31+and CD34+vessel and aberrantly branched vasculature were reduced in the tumor in the RT+HPTA group as compared to the other groups,indicating that tumor angiogenesis was inhibited and the integrity of the vasculature was improved.These findings suggested that combining HPTA with RT can inhibit tumor neovascularization and promote vascular normalization,such effect may be associated with the secretion of IFN-γ by anti-tumor Ml macrophages.Conclusions1.Radiotherapy alone can not cause abscopal effect,HPTA can effectively promote the occurrence of abscopal effect of radiation.2.HPTA can regulate the polarization of tumor-associated macrophages to anti-tumor M1 type in unirradiated tumor tissues and promote the secretion and release of pro-inflammatory cytokines,showing pro-inflammatory effects.3.HPTA can effectively inhibit tumor angiogenesis and promote vascular normalization in unirradiated tumor tissues,and the mechanism may be related to IFN-γ.