Abstract 60: An emerging paradigm of Cxcl12 & Cxcr4 involvement in breast cancer metastasis
Authors: Anastasiadou Dimitra P. , Sanchez Luis R. , Duran Camille L. , Burt Joseph , Chen Xiaoming , Lin Yu , Eddy Robert , Harney Allison S. , Entenberg David , Condeelis John S. , Oktay Maja H. , Karagiannis George S. 
Published: April 2023

Abstract
The Cxcl12/Cxcr4 signaling axis has been shown to promote metastasis in multiple mouse models of breast carcinoma, and has been linked to breast cancer cell seeding, homing, survival, and proliferation at future metastatic sites. The precise mechanism by which Cxcr4+ breast cancer cells escape the primary tumors (which also highly express Cxcl12), remains poorly understood. By using a novel multichannel immunofluorescence (mIF) based methodology for quantifying chemotactic gradients in fixed tissue, we here demonstrate that Cxcl12 gradients in mouse primary breast tumors are concentrically expressed around sites of cancer cell intravasation known as Tumor Microenvironment of Metastasis (TMEM) doorways. Via distance analysis algorithms, we additionally demonstrate that TMEM doorway-mediated Cxcl12 gradients associate with Cxcr4+ breast cancer cells migrating towards the underlying TMEM doorways. Consistent with this observation, pharmacological inhibition of the Cxcl12/Cxcr4 pathway significantly abrogates the translocation of Cxcr4+ cancer cells to TMEM doorways, suppressing TMEM doorway-mediated metastatic dissemination. However, targeted elimination of the Cxcr4 gene specifically from breast cancer cells, paradoxically results in a suboptimal response, thus suggesting the existence of a bypass or compensatory mechanism. Previously, it was shown that Cxcr4+ tumor-associated macrophages (TAMs) support the invasive and migratory properties of tumor cells that utilize TMEM doorways. We thus hypothesized that, absent Cxcr4 expression in tumor cells, accompanying Cxcr4+ TAMs may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, clodronate-mediated TAM depletion results in the significant suppression of Cxcr4+ cancer cell translocation to TMEM doorways and their subsequent dissemination to the peripheral circulation and future metastatic sites. Finally, we used a variety of stromal and immune cell lineage markers to identify the precise source of TMEM doorway-generated Cxcl12 gradients in mouse primary breast cancers. Despite the fact that blood vessels (irrespective of presence of TMEM doorways) were primarily lined by Pdgfrb+ stromal cells with basal Cxcl12 expression, TMEM-generated Cxcl12 gradients were specifically linked to a subset of Cxcl12+Iba1+ perivascular TAMs. Pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not significantly affect Cxcl12/Cxcr4- mediated translocation of Cxcr4+ tumor cells to TMEM doorways. Overall, our data support a new paradigm implicating the Cxcl12/Cxcr4 axis during the early stages of the metastatic cascade, and point to a new avenue for rationalized antimetastatic treatments for breast cancer.
Citation Format: Dimitra P. Anastasiadou, Luis R. Sanchez, Camille L. Duran, Joseph Burt, Xiaoming Chen, Yu Lin, Robert Eddy, Allison S. Harney, David Entenberg, John S. Condeelis, Maja H. Oktay, George S. Karagiannis. An emerging paradigm of Cxcl12 & Cxcr4 involvement in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 60.