| Objective:Neoadjuvant chemotherapy(NACT)plays an indispensable role in the treatment of breast cancer(BC).The response to NACT differs among individuals.Pathological complete response(pCR)is a significant predictor of overall survival and disease-free survival in BC,and approximately 20-30%of patients achieve pCR after NACT.However,certain risk factors are associated with the development of chemotherapy resistance.Therefore,early prediction of the pathological response to NACT is critical,and it may help optimize individual chemotherapeutic strategies in BC patients.The tumor response to NACT is determined by intricate interactions between tumor cells and the surrounding microenvironment.Ki-67 is a nuclear antigen that is widely used as a marker of cellular proliferation.Ki-67 expression before NACT is a clinical predictor of NACT responses in BC.However,the predictive value of a single indicator remains controversial.Angiogenesis,as an important factor in the tumor microenvironment,is considered a rate-limiting step in BC progression.Dynamic optical breast imaging(DOBI)is a noninvasive technique based on the use of near-infrared light to detect relative blood-volume and oxygen saturation variations in the microvascular bed of biological tissues.Blood perfusion variations evaluated by optical imaging show good predictive power regarding different responses to NACT in BC patients,although more research is needed.The extracellular matrix,as anther important factor in the tumor microenvironment,also plays an important role in the response to NACT.Shear-wave elastography(SWE)is a non-invasive ultrasound imaging method that can quantify tissue stiffness in vivo.High stiffness on SWE is associated with chemotherapy resistance in BC,although this finding has been reported in relatively few studies.Tumor stiffness,blood flow characteristics,and Ki-67 expression are early predictors of the pathological response to NACT.However,to the best of our knowledge,these parameters have not been directly compared to date.Recent studies show that high matrix stiffness could induce epithelial to mesenchymal transition(EMT)and tumor progression by activating Twist family BHLH transcription factor 1(TWIST-1).In addition,TWIST-1 over-expression is associated with short survival and a poor response to chemotherapy in patients with cancer.Hypoxia-inducible factor 1-alpha(HIF-1α),which mediates adaptation to hypoxia in cells,has been shown to be associated with matrix stiffness.Activation of the hypoxia pathway by HIF-1α contributes to the development of radiotherapy and chemotherapy resistance.A recent study showed that β1 integrin(ITGB-1)plays a pivotal role in the regulation of matrix stiffness,and ITGB-1 expression is associated with chemoresistance and metastasis in BC.Most of the studies cited above were based on basic experiments in vitro or in animal models.However,no study has investigated the relationships between HIF-1α,TWIST-1,and ITGB-1 expression and tumor stiffness in BC patients,or the predictive diagnostic performance of these biomarkers for predicting NACT responses.The present study was performed by the following three parts:Firstly,we examined the patients of BC treated with NACT to compare the ability of tumor size,tumor stiffness,optical imaging features and Ki-67 expression alone or in combination to predict the response to NACT in BC.Secondly,we analyzed BC patients received NACT to determine the association of HIF-1α,TWIST-1,and ITGB-1 expression with tissue stiffness,oxygen score(OS),and pathological responses.In addition,we investigated the power of HIF-1α,TWIST-1,and ITGB-1 alone or in combination with Ki-67 to predict the response to NACT in BC.Finally,a nude mouse of BC xenografts was prepared and treated with hyaluronidase to reduce the tumor stiffness.Three cycles of NACT were performed to mices.We analyzed the effect of lower tumor stiffness on the blood volume and oxygen saturation of local tumor tissue,then the relationships between the key proteins HIF-1α,TWIST-1,ITGB-1,VEGF,CD31 expression and pathological response to NACT were also investigated.The purpose of our study was to explore the initial mechanism about how the tumor stiffness predicts the NACT response.Method:1、Efficacy of gray-scale ultrasound,shear-wave elastography,dynamic optical breast imaging and traditional predictor Ki-67 for predicting the pathological response to neoadjuvant chemotherapy in breast cancer Between March 2014 and May 2020,the prospective study recruited patients with core needle biopsy confirmation of invasive breast cancer consecutively,who also fulfilled the inclusion criteria.Tumor size,tumor stiffness(maximum stiffness(Emax),mean stiffness(Emean)),blood score(BS),oxygen score(OS)were evaluated before(t0),during(t1-t5),and at the end of NACT(t6)by gray-scale ultrasound,shear-wave elastography and optical imaging,respectively.The relative changes about these imaging parameters after the first and second NACT cycles were recorded asΔSize(t1/t2),ΔE(t1/t2),ΔBS(t1/t2)and ΔOS(t1/t2).Ki-67 expression was quantitatively evaluated by immunohistochemistry using core biopsy specimens obtained before NACT.Pathological responses were evaluated by residual cancer burden(RCB).The predictive power of tumor size,SWE stiffness,BS,OS and Ki-67 was compared using receiver operating characteristic curves(ROC)and the Z-test.Finally,a new prediction model PredRCB1(predicted RCB scores 1)was developed by combining the predictors with the largest AUC(SWE stiffness,BS and OS)and the traditional marker(Ki-67)according to the results of the multivariable linear regression model.Subgroup analysis was conducted to investigate the predictive power of PredRCBl for different outcomes according to different characteristics.2、Evaluation of the correlation of HIF-1α,TWIST-1 and ITGB-1 with tumor stiffnessm,and the peformance for predicting response to neoadjuvant chemotherapy in breast cancer Between February 2014 and July 2019,patients with core needle biopsy confirmation of invasive BC that fulfilled the inclusion criteria were included in the study by consecutively.Tumor stiffness and oxygen score(OS)were evaluated before NACT by shear-wave elastography and optical imaging;HIF-1α,TWIST-1,ITGB-1,and Ki-67 expression was quantitatively assessed by immunohistochemistry of tumor samples obtained by core needle biopsy.Pathological responses were evaluated by RCB.Indexes were compared among different RCB groups,and associations of HIF-1α,TWIST-1,ITGB-1,and Ki-67 with tumor stiffness and OS were examined.The value of HIF-1α,TWIST-1,ITGB-1,and Ki-67 for predicting NACT responses was assessed by ROC curves.Finally,a new prediction model PredRCB2(predicted RCB scores 2)was combined with the largest AUC of new predictors(HIF-1α,TWIST-1,and ITGB-1)and the traditional one(Ki-67)according to the results of the multivariable linear regression model.Subgroup analysis was conducted to investigate the predictive power of PredRCB2 for different outcomes according to different characteristics.3、Effect of the tumor matrix stiffness on the pathological response to neoadjuvant chemotherapy and its related intial mechanisms Three-negative breast cancer cells,MDA-MB-231,were cultured to the logarithmic growth phase,then the cells were collected to prepare a nude mouse xenograft model.The nude mice of tumor model were randomly divided into four groups(n=10):The control group,in which mice were treated with the same amount of normal saline;The Hyaluronidase group,in which mice were treated with Hyaluronidase on the zero day,3rd day,6th day;The NACT group,in which mice were treated with chemotherapy drug on the 1st day,4th day and 7th day;The combined intervention group,in which mice were treated by hyaluronidase on the zero day,3rd day,6th day and chemotherapy drug on the 1st day,4th day and 7th day.Tumor volume was measured and calculated with every three days.On the zero day,3rd day,6th day,and 9th day,SWE and DOBI were performed to detect the tumor stiffness,BS and OS in each group.At the end of the experiment,the nude mice were sacrificed by cervical dislocation,and the levels of HIF-1α,TWIST-1,ITGB-1,VEGF and CD31 were detected by immunohistochemistry and western Blot.Results:1、Part one A total of 145 patients who met the inclusion criteria were finally enrolled.(ⅰ)Δ Size(t2),E2mean,ΔEmean(t2),BS2,OS2 and Ki-67 had a greater power than other indexes for predicting a favorable response(AUC=0.62,0.78,0.82,0.81,0.78,0.81)and a resistance response(AUC=0.64,0.80,0.85,0.79,0.78,0.84).The Z-test of AUCs indicated that there were significant differences in AUC s for Δ Size(t2)and other parameters(P<0.05).Whereas,there were no significant differences in AUCs for ΔEmean(t2),E2mean,BS2,OS2 and Ki-67(P>0.05).(ⅱ)PredRCBl which combined ΔEmean(t2),BS2 and Ki-67 had better predictive performance than any parameter alone for a favorable response(AUC=0.90)and resistance(AUC=0.93).(ⅲ)PredRCB1 showed good predictive ability in the different subgroups.2、Part two A total of 104 patients who met the inclusion criteria were finally enrolled.(ⅰ)Higher tumor stiffness was strongly correlated with higher HIF-1α,TWIST-1,and ITGB-1 expression and lower OS.(ⅱ)HIF-1α,TWIST-1,ITGB-1,and Ki-67 expression exhibited good and similar diagnostic performances for predicting a favorable response(AUC=0.81,0.85,0.79,0.80)and resistance to NACT(AUC=0.83,0.86,0.84,0.85).(ⅲ)PredRCB2 which combined TWIST-1 and Ki-67 had better predictive performance than any parameter alone for a favorable response(AUC=0.88)and resistance(AUC=0.92).(ⅲⅰ)PredRCB2 showed good predictive ability in the different subgroups.3、Part three The BC xenografts nude-mouse model was successfully prepared.We found that hyaluronidase treatment could significantly reduce tumor matrix stiffness;The reduction of tumor matrix stiffness could increase the oxygen content,inhibit the expression of HIF-1α,TWIST-1,ITGB-1,VEGF,CD31,which could also promote the sensitivity of tumors response to chemotherapy.Conclusion:Firstly,for the imaging,tumor SWE stiffness,optical imaging features,and Ki-67 expression exhibited good and similar performances for the early identification of NACT responses in BC and were significantly superior to that of tumor size.Futhermore,this study highlights the potential utility of PredRCB1,which is the combination of the tumor stiffness,blood flow characteristics and Ki-67 expression,improving the predictive ability of each single indicator.Secondly,from the perspective of molecular biology,the present study is the first to comfirmed that higher HIF-1α,TWIST-1,and ITGB-1 expression levels were strongly correlated with higher tumor stiffness and lower OS in BC patients.Moreover,HIF-1α,TWIST-1,and ITGB-1 expression exhibited a good diagnostic performance for the early prediction of NACT responses in BC.In addition,this study highlighted the potential value of PredRCB2,which is the combination of the TWIST-1 and Ki-67 expression,improving the diagnostic performance of single markers for the early prediction of different pathological responses.PredRCB1 and PredRCB2 showed good predictive ability in the overall population and subgroups of patients,which may help tailor individualized treatment regimens for BC patients receiving NACT.Finally,our results demonstrated that the tumor stiffness was reduced through the degradation of hyaluronic acid under hyaluronidase treatment in nude mice models of breast cancer xenografts.Under hyaluronidase treatment,the blood volume was decreased and the oxygen saturation in local tumor tissues were increased;the expression of HIF-1α,TWIST-1,and ITGB-1 was down-regulated;the sensitivity of the tumor to chemotherapy was significantly increased.The relationship between tumor stiffness,hypoxia and chemoresistance was further confirmed in mice models.This study provided an initial experimental basis for further exploration the novel working hypothesis "high tumor stiffness-tissue hypoperfusion hypoxia-HIF-1α/TWIST-1/ITGB-1 activation-resistance to NACT". |
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