The Effects And Mechanisms Of Immune Checkpoint Therapy On Tumor Blood Vessels

BackgroundImmune checkpoint blockade(IC B),such as cytotoxic T-lymphocyte-associated protein 4(CTLA4)or programmed cell death 1(PD1),has demonstrated long-term therapeutic benefits in multiple cancer types,including melanoma,non-small cell lung cancer,renal cell carcinoma,however only in about one-quarter of cancer patients.The underlying mechanism for this selective therapeutic efficacy remains unclear.Moreover,there’s no reliable method to predict the efficacy of immune checkpoint inhibitors.Hypothesis and aimsAccording to these facts,we hypothesize that both T cell activation and tumor microenvironment(TME)remodeling are required for immune checkpoint blockade therapy to cure a cancer.This hypothesis will be tested by the following specific aims.Aim 1: to eastablish ICB-sensitive and-resistant tumor models.Aim 2: to understand the mechanism of selective therapeutic efficacy of immune checkpoint therapy.Aim 3: to identify reliable and noninvasive biomarkers to predict ICB responsiveness.MethodsIn orthotopic(EO771,4T1,and MCa P0008)and spontaneous(MMTV-Py VT)breast tumor models,as well as colon tumor models(MCa38 and CT26),(1)we measured the changes of tumor volume and weight after ICB or isotype control treatments;(2)we analyzed the effects of ICB on tumor blood vessels by using immunohistochemical staining;(3)we quantified the m RNA levels of angiogenic and angiostatic factors after IC B therapy using real-time PCR;(4)we analyzed the quantities and phenotypes of tumor-infiltrating immune cells after ICB therapy using a flow cytometry;(5)we analyzed the effects of in vivo depletion of CD4+ and CD8+ T cells on tumor growth and tumor vessels;(6)we analyzed the effect of ICB therapy on IFN-γ production in T cells through intracellular staining;(7)we determined the roles of IFN-γ in C TLA4 blockade therapy through IFN-γ neutralization or using IFN-γ receptor knockout mice;(8)we tried to identify reliable and noninvasive biomark ers for predicting ICB-responsiveness;(9)we analyzed the time course influence of ICB therapy on tumor vascular function and tumor growth;(10)we measured the changes in tumor vascular perfusion upon anti-CTLA4 therapy by using color doppler angiography and the 3D imaging.Results(1)The tumor growth data suggested that the orthotopic EO771 and genetically engineered MMTV-Py VT spontaneous breast tumor,as well as MCa38,CT26 colon tumor models were ICB-sensitive,whereas the MCa P0008 and 4T1 breast tumor models were ICB-resistant.(2)ICB therapy reduced tumor vessel density,increased pericyte coverage,enhanced vessel perfusion,and reduced tissue hypoxia in treatment-sensitive,but not in treatment-resistant,tumor models,suggesting that ICB therapy induces tumor vascular normalization.Moreover,IC B therapy increased vessel perfusion(IVP)in ICB-responders compared with those in the isotype control group or non-responders.In addition,anti-CTLA4 therapy didn’t affect the pattern and function of blood vessels in normal tissue.(3)IC B therapy did not alter the transcription of comventional pro-angiogenic factors,whereas ICB therapy up-regulated the levels of angiostatic factors IFN-γ and CXCL9.(4)ICB treatment increased the proportions of tumor-infiltrating CD8+ T cells.(5)Simultaneous depletion of CD4+ and CD8+ T cells completely negated the IVP phenotype and the inhibition of tumor growth induced by anti-C TLA4 therapy,which was similar to C D8+ T cell depletion.Howerver,depletion of CD4+ T cells alone increased tumor vessel perfusion and inhibited tumor growth,which was comparable to anti-CTLA4 therapy.(6)Intracellular staining analysis showed that anti-CTLA4 therapy elevated IFN-γ expression in CD8+ T cells.(7)In vivo neutralization of IFN-γ or IFN-γ receptor deficiency abolished the effects of anti-CTLA4 therapy on vessel perfusion and tumor growth.These findings suggest that IFN-γ signaling is required for the vascular remodeling and tumor growth inhibition induced by anti-CTLA4 therapy.(8)In sensitive tumor models,increased vessel perfusion by ICB therapy correlates with its antitumor efficacy.Thus,individual EO771 tumors could be stratified as ‘responders’ or ‘non-responders’ according to changes in tumor blood vessel perfusion.(9)The time-course study suggested that anti-CTLA4 therapy increases vessel perfusion before noticeable tumor size changes in EO771 tumors.(10)By using three-dimensional ultrasonography and color Doppler measurement to estimate tumor volume and the percentage of perfused tumor blood vessel volume,the data showed that global changes in tumor vessel perfusion by IC B could be measured noninvasively by radiological methods to predict individual responses to ICB therapy.Conclusions and SignificanceImmune checkpoint blockade induces tumor vascular normalization via the accumulation of CD8+ T cells and the production of IFN-γ.ICB increases tumor vessel perfusion prior to detectable changes in tumor volume.Furthermore,IVP by ICB correlates with its antitumor efficacy.These findings suggest that IVP could be served as a noninvasive biomarker to predict individual responses to ICB therapy.Based on these results,we proposed a new mechanism of immune checkpoint therapy: immune checkpoint inhibitors activate C D8+ T cells to normalize tumour blood vessels.Vascular normlaization remodels the TME and alleviates immunosupportion.This in turn facilitates the expansion and activation of C D8+ T cells,resulting in more vascular and TME remodelling,which ultimately leads to long-term tumour control.This new mechanism could explain the selective efficacy of ICB.Moreover,this study,for the first time,suggests that alteratiions in tumor blood vessel perfusion could be used to predict ICB-efficacy.Given that vascular perfusion can be monitored in real time by noninvasive radiologic methods,it is conceivable that IVP could s ignificantly upgrade the current practice of cancer immunotherapy,to achieve genuinely personalized cancer immunotherapies to benefit cancer patients.