Abstract P5-13-05: Multiplex spatial systems analysis of responses to spatially separate nanoliter doses of drug predicts systemic immune-modulating combination treatments in breast cancer
Authors: Tatarova Zuzana , Blumberg Dylan C , Coussens Lisa M , Mills Gordon B , Jonas Oliver , Gray Joe W 
Published: March 2022

Abstract
Most targeted and chemotherapeutic agents in use today affect both the tumor and the associated tumor microenvironment (TME) and should be combined to achieve effective control by simultaneously attacking tumor intrinsic vulnerabilities, enhancing immune surveillance, and mitigating stromal mediators of resistance. We have developed an integrated technological platform that enables rapid identification of such combinations by effective, fast and harmless assessment of TME responses to multiple drugs or drug combinations. This approach uses an implantable microdevice for delivery of dozens of treatments into spatially separated regions of a single living tumor and multiplexed histology analyses with probes for 30+ proteins to interrogate the responses of tumor epithelial cells, cancer associated fibroblasts, endothelial cells, lymphoid and myeloid immune lineages. Responses of interest include changes in differentiation state, antigenicity and immunogenic cell death. We demonstrated the utility of this platform by performing an unbiased in vivo screen to assess the effects of seven FDA approved drugs and combinations thereof on two late-stage mouse models of breast cancer with intact immunity. These studies indicated that the most effective, immunogenic, tumor cell kill was produced by the epigenetic modulator, panobinostat, and was linked with upregulation of MHC-I expression on tumor cells, and recruitment of antigen presenting macrophages and cytotoxic neutrophils. Increased expression of Galectin-3 and Neuropilin-1 on cancer stem cells and neutrophils, respectively, correlated with the therapeutic response three days after drug exposure, suggesting a functional role of these two proteins in early events of induced anti-tumor immunity. Our spatial systems analyses also revealed a key role for density of cancer stem cells (CSCs) and formation of fibroblasts/ECM barriers as potential resistance mechanisms. Addition of the BCL2 inhibitor, venetoclax, enhanced panobinostat anti-tumor activity, and the local phenotypic cell responses involving dendritic cell recruitment and clustering to CSC microculture suggested the use of anti-CD40 immunotherapy to induce maximum therapeutic effect. We demonstrated that a panobinostast-venetoclax-anti-CD40 therapeutic combination predicted from microdevice analyses was effective in whole animal studies in multiple models of breast cancer and often resulted in complete tumor control. Generalization of this approach promises to enable rapid design of personalized drug combination regimens that maximize tumor cell eradication by direct action on the tumor cells and by creating effective antitumor microenvironments. We demonstrated the utility of the approach in model systems but application in humans is now underway.
Citation Format: Zuzana Tatarova, Dylan C Blumberg, Lisa M Coussens, Gordon B Mills, Oliver Jonas, Joe W Gray. Multiplex spatial systems analysis of responses to spatially separate nanoliter doses of drug predicts systemic immune-modulating combination treatments in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-05.