Cancer-associated fibroblasts (CAFs) play important roles in breast cancer (BC) progression and metastasis. Here we investigated whether CAFs from indolent vs. aggressive BCs differ in gene expression profiles and how they impact metastasis. Genotypic differences in CAF lines from basal-like (CAF23BAS) and luminal-A BC (CAF19LA), were compared and effects on CAF-induced phenotypes of estrogen receptor (ER) positive BC models evaluated. Co-injection of MCF7 with CAF23BAS cells enhanced tumor metastasis in vivo, while CAF19LA did not. CXCL12 was strongly overexpressed in CAF23BAS. BC cells isolated from MCF7 + CAF23BAS tumors were enriched in epithelial-mesenchymal transition (EMT) genes and cancer stem cell (CSC)-like behavior. Chronic CXCL12 exposure in vitro, as may occur in BC with high CXCL12-secreting CAFs, phenocopied CAF23BAS-enhanced metastasis. Single cell analysis of primary human BC revealed CAFs are the major source of CXCL12 in breast tumors. A high CXCL12-CAF gene expression profile was prognostic of poor BC outcome and was strongly over-represented in CAFs within BC metastases. Finally, gene expression changes induced in MCF7 cells by co-injection with CAF23BAS in vivo correlated significantly with gene expression differences between normal and malignant epithelial cells in BC containing high CXCL12 CAFs. These findings suggest that CXCL12 overexpressing CAFs can induce gene expression changes in breast cancer that promote breast cancer metastasis, potentially through expansion of the CSC population. Targeting the CAF CXCL12/CXCR4 axis may offer a novel treatment strategy for metastatic breast cancer and warrants further investigation. Cancer associated fibroblasts (CAF) within the breast tumor microenvironment influence breast cancer behavior. Our study indicates that high CXCL12-expressing CAFs can induce a stable metastatic phenotype in estrogen receptor positive breast cancer models. Gene expression similarities between a high CXCL12 CAF line and high CXCL12-expressing CAFs from primary and metastatic human breast cancers define a CXCL12-high CAF signature that is prognostic of poor BC patient outcome. Furthermore, gene expression changes induced in MCF7 cells by CXCL12 high CAFs in vivo were similar to the gene expression differences between normal and malignant breast epithelial cells in breast cancers containing CXCL12 high CAFs. Disruption of CAF-driven, CXCL12-mediated reprogramming of breast cancer cells might provide an opportunity to prevent or treat breast cancer metastasis.

Circulating tumor cells (CTCs), a population of cancer cells that represent the seeds of metastatic nodules, are a promising model system for studying metastasis. However, the expansion of patient-derived CTCs ex vivo is challenging and dependent on the collection of high numbers of CTCs, which are ultra-rare. Here we report the development of a combined CTC and cultured CTC-derived xenograft (CDX) platform for expanding and studying patient-derived CTCs from metastatic colon, lung, and pancreatic cancers. The propagated CTCs yielded a highly aggressive population of cells that could be used to routinely and robustly establish primary tumors and metastatic lesions in CDXs. Differential gene analysis of the resultant CTC models emphasized a role for NF-κB, EMT, and TGFβ signaling as pan-cancer signaling pathways involved in metastasis. Furthermore, metastatic CTCs were identified through a prospective five-gene signature (BCAR1, COL1A1, IGSF3, RRAD, and TFPI2). Whole-exome sequencing of CDX models and metastases further identified mutations in constitutive photomorphogenesis protein 1 (COP1) as a potential driver of metastasis. These findings illustrate the utility of the combined patient-derived CTC model and provide a glimpse of the promise of CTCs in identifying drivers of cancer metastasis.

Circulating tumor cells (CTCs), a population of cancer cells that represents the seeds of metastatic nodules, are a promising model system for studying metastasis. However, expansion of patient-derived CTCs ex vivo is challenging and dependent on the collection of high numbers of CTCs, which are ultra-rare. Here, we report the development of a combined CTC and CTC-derived xenograft (CDX) platform for expanding and studying patient-derived CTCs from metastatic colon, lung, and pancreatic cancers. Propagated CTCs yielded a highly aggressive population of cells that could be used to routinely and robustly establish primary tumors and metastatic lesions in CDXs. Differential gene analysis of the resultant CTC models emphasized a role for NF-kB signaling as a pan-cancer signaling pathway involved in metastasis. Furthermore, metastatic CTCs were identified through a prospective 5-gene signature ( BCAR1, COL1A1, IGSF3, RRAD, and TFPI2 ). Whole-exome sequencing of CDX models and metastases further identified mutations in constitutive photomorphogenesis protein 1 ( COP1 ) as a potential driver of metastasis. These findings illustrate the utility of the combined patient-derived CTC model and provide a glimpse of the promise of CTCs in identifying drivers of cancer metastasis.

Background: Metastatic disease is the foremost cause of breast cancer (BC) related mortality in women. One of the crucial challenges in reducing metastasis-related mortality is in identifying and understanding why certain BCs metastasize and recur. Although cancer cell-intrinsic factors are a key determinant in BC metastasis, host-intrinsic factors like the cells of the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs) are what might be driving certain BCs to metastasize and recur. We recently showed that CAFs from the primary (TME) enter into circulation as cCAFs, form heterotypic clusters with tumor cells, and arrive at metastatic sites. This interaction between CAFs and tumor cells is crucial in furthering the metastatic cascade. However, the functional heterogeneity of the different CAF phenotypes and how that impacts BC metastasis is unknown. Stromal derived factor-1 (SDF-1/CXCL12) is an important chemokine that is known to be involved in promoting tumor cell invasion and metastasis. In this study, we examine the functional differences between CAF isolated from different molecular subtypes of BC. Furthermore, we elucidate the role of CAF secreted SDF-1 as being one of the mechanisms by which a subset of CAF cells permanently reprograms poorly-metastatic MCF-7 cells to augment EMT-driven genes and become metastatic in vivo. Methods: We used primary CAF cell lines derived from BCs of different molecular subtypes and examined their global gene expression profiles using RNAseq. We used NSG mice xenografted with MCF-7 BC cells and primary CAF cells to evaluate the contribution of two different CAF phenotypes in promoting BC progression and metastasis. We developed dissociated tumor cell lines from CAF co-injected MCF-7 xenografts to determine the molecular changes that CAFs impart to MCF-7 cells in vivo. Using limiting dilution tumor xenograft assays and gene expression profiles we examined the molecular pathways, BC stemness markers, and tumor-initiating capacity of the dissociated tumor cell lines. By long-term SDF-1 treatment of MCF-7 cells in vitro and using these cells in various RNASeq, in vitro, and in vivo assays we determined the role of SDF-1 in altering the phenotype of MCF-7 cells. Results: We found that CAFs from different molecular subtypes have a differential effect on tumor initiation, progression, and metastasis in xenograft assays. We also found that a subset of CAFs has the ability to make the poorly-metastatic MCF-7 cells metastatic in vivo. We also found that this subset of CAF cells has a higher expression of SDF-1 and the tumors formed with the co-injection of MCF-7 cells with this subset of CAF cells are enriched in cancer stem cell-like metastasis initiating cells. We also found that upon serial transplant, the tumors formed by CAF-reprogramed MCF-7 cells have a gene expression profile that shows enrichment for EMT-driven genes and these tumors are robustly metastatic. Furthermore, we found that the SDF-1/CXCR4 axis is one of the critical mechanisms by which this subset of CAF cells permanently reprograms the phenotype of the MCF-7 cells. Conclusions: CAFs are a highly heterogeneous population of cells in the breast TME. This study examines the contribution of the functional heterogeneity of the CAF cells in BC metastasis. We have found that a unique subset of CAF cells has the ability to reprogram a poorly metastatic BC cell to not only become metastatic but induce a permanent EMT-driven phenotypic shift in the BC cells. SDF-1 may be one of the mechanisms that drive this CAF-induced change and understanding the CAF functional landscape and its role in BC metastasis might be crucial to developing new therapeutic modalities to abrogate BC metastasis. Citation Format: Utsav Sharma, Jun Sun, Kelsie Medina-Saenz, Susan Bare, Philip Miller, Manuel Picon-Ruiz, Joyce Slingerland, Dorraya El-Ashry, Marc Lippman. Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis [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 P5-06-08.

Abstract Background: Cancer associated fibroblasts (CAFs) are the most abundant non-cancerous cell component of the breast cancer (BC) TME and have been shown to participate in most all steps of metastasis. We have previously shown that CTCs cluster with circulating CAFs (cCAFs) in BC patients at all disease stages, and this observation is recapitulated in the mouse PYMT BC model. Our central hypothesis is that cCAF/CTC co-clusters comprise the functional metastatic unit, and here we sought to determine the role of cCAFs in co-clusters on CTC extravasation and metastatic seeding. Methods: Xenografts: In vitro co-clusters were produced by culturing human CAF23 cells and MDA231(Ffluc) cells in ULA plates for 18 hrs. Clusters were injected via tail vein into NSG mice and metastatic growth was monitored by BLI. Endothelial binding assays: In vitro clusters, single CAFs or single BC cells were labeled with Cell Tracker dyes, pipetted onto confluent HUVEC monolayers, and allowed to bind for 30 minutes before gentle washing. Bound cells were counted on a fluorescent microscope. Ex vivo extravasation assays: Cell Tracker labeled co-clusters were injected via tail vein into FVB mice. One hour before sacrifice, FITC-lectin was injected via tail vein to label lung endothelia. Lungs were then extracted and imaged by confocal microscopy. Results/Discussion: Xenograft experiments demonstrate that within the first week, CAF23/MDA231 co-clusters produce durable, proliferative lung metastases faster than MDA231 monoclusters. This early difference in metastatic growth suggests that CAFs help BC cells establish metastases faster, possibly by aiding extravasation into the lung parenchyma. Using our ex vivo model of lung extravasation, we saw that significantly more CAF23/MDA231 co-clusters than MDA231 monoclusters were in the process of and/or had extravasated into the lung parenchyma at 8 and 24 hrs post-injection. In vitro binding experiments revealed that HUVEC endothelial cells bind more CAF23/BC co-clusters than BC monoclusters. Similarly, HUVEC cells bound significantly more single CAF23 or CAF19 cells than BC cells. CAF binding increased when endothelial e-selectin expression was induced with IL1-b; binding to e-selectin being the first step in cancer cell rolling and extravasation. Immunofluorescence and flow cytometry showed that CAFs express the CA19-9 antigen, sialyl-Lewis A (sLeA), which acts as an e-selectin ligand. Tannic acid, an inhibitor of the chemokine SDF1/CXCL12, decreased binding, as did 5mM EDTA. CD44 siRNA had no effect on binding whereas depletion of hyaluronin, a ligand of CD44, had a significant effect on CAFs but not on BC cells. Together, these experiments point to CAFs in co-clusters as critical enhancers of endothelial binding and extravasation, which may ultimately result in faster establishment of metastases and enhanced survival of CTCs. Future investigations will focus on therapeutic targeting of the mechanisms we have identified to block cCAF/CTC extravasation and metastasis. Citation Format: Angela Spartz, Benjamin Troness, Utsav Sharma, Geneva Taylor, Dorraya El Ashry. Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO038.

Abstract Introduction: Breast cancer metastasis is a multi-step process that involves cellular migration, invasion, travel through the circulation, extravasation and secondary site proliferation. Circulating tumor cell (CTC) travel is thought to be via rolling on vascular endothelium, and CTC clusters have been shown to have a shorter circulation half-life than single CTCs due to increased entrapment within capillaries. We have previously shown that CTCs cluster with circulating cancer associated fibroblasts (cCAFs) in breast cancer patients and this observation is recapitulated in the mouse PYMT breast cancer model. Circulating CAF/CTC clusters are seen in patients of all stages and in all PyMT mice with tumors, before overt metastases. Our central hypothesis is that cCAF/CTC co-clusters comprise the functional metastatic unit and here we sought to determine the role of cCAFs in co-clusters on metastatic seeding. Methods: Xenografts: In vitro co-clusters were produced by culturing 75,000 human CAF23 cells and 75,000 MDA231(Ffluc) cells in ultra-low attachment plates for 18 hrs. Clusters were injected via tail vein into NSG mice and metastatic growth was monitored by bioluminescent imaging. Endothelial binding assays: In vitro clusters were made as for xenografts except that CAFs and BC cells were labeled with Cell Tracker dyes. Clusters were pipetted onto confluent HUVEC monolayers and allowed to bind for 30 minutes before gentle washing. Bound cells were counted on a fluorescent microscope. Ex vivo extravasation assays: Co-clusters were made as above with Cell Tracker labeled MDA231 and CAF23 cells and injected via tail vein into FVB mice. Lung endothelia was labeled by tail vein injection of FITC-lectin an hour before lungs were extracted and imaged by confocal microscopy. Results: Xenograft experiments demonstrate that within the first week, CAF23/MDA231(Ffluc) co-clusters produce durable, proliferative lung metastases faster than MDA231(Ffluc) monoclusters. This early difference in metastatic growth suggests that CAFs help cancer cells establish metastases faster, possibly by enhancing the binding and rolling of CTCs along the vascular endothelium and/or by aiding extravasation into the lung parenchyma. In vitro binding experiments revealed that CAF23/MDA231 co-clusters bind to HUVEC endothelial cells better than MDA231 monoclusters under static conditions. Similar results were observed with the aggressive basal cell line, DT28. To determine the effect of shear stress, we performed endothelial binding assays in an orbital shaker at 100 rpm (used to mimic oscillatory and continuous flow shear stress), and under these conditions, CAFs continue to bind to HUVECs at significantly higher numbers than MDA231 cells. CAF binding increased two-fold when endothelial e-selectin expression was induced with IL1-β. Immunofluorescence and flow cytometry showed that CAFs express the CA19-9 antigen, sialyl-Lewis A (sLeA), which acts as an e-selectin ligand; binding to e-selectin being the first step in cancer cell rolling and extravasation. Using our ex-vivo extravasation imaging protocol, we observed that significantly more CAF23/MDA231 co-clusters than MDA231 monoclusters were in the process of extravasation and/or had extravasated into the lung parenchyma at 8 and 24 hrs post-injection. Conclusions: Together, these experiments point to CAFs in co-clusters as critical enhancers of endothelial binding and extravasation, which may ultimately result in quicker establishment of metastases and enhanced survival of CTCs in clusters. Future studies will focus on identifying the molecular mechanisms in these processes facilitated by cCAFs that could be amenable to therapeutic targeting. Citation Format: Angela Spartz, Benjamin Troness, Utsav Sharma, Dorraya El-Ashry. Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-28.

Abstract Background: Cancer associated fibroblasts (CAFs) help form the tumor microenvironment and we have demonstrated that circulating CAFs (cCAFs) from primary tumors are found in breast cancer patients and mouse models. cCAFs form heterotypic co-clusters with circulating cancer cells (CTCs) that exhibit enhanced metastatic seeding potential. CTC/cCAF co-clusters appear in the early stages of disease before the appearance of overt metastases. We have shown that the ability of CTCs and CAFs to form co-clusters is linked to the CTCs' intrinsic metastatic capacity, particularly the cancer stem cell (CSC)-like phenotype. Metastatic breast cancer cells, such as MDA-MB-231s and DT28s, readily form co-clusters with CAFs in vivo and in vitro while non-metastatic breast cancer cells, such as MCF7s, do not. CD44 is a cell surface adhesion receptor commonly associated with the CSC phenotype. CD44 is abundantly expressed by CAFs and binds a number of ligands with established roles in cancer, including hyaluronic acid (HA) and osteopontin. Here, we demonstrate that CD44 and its primary ligand, HA, mediate the co-clustering of CTCs and CAFs. Methods: MDA-MB-231 and DT28 (Primary TNBC) cells were treated with CD44 siRNA to knock down CD44 expression. Control and siRNA-treated MDA-MB-231 or DT28 cells were seeded in ultra-low attachment plates with CAF23s (Primary CAFs from TNBC). After 24 hours, cells were captured on microfilters using a faCTChecker microfluidic filtration instrument (Circulogix). Cancer cells and CAFs were stained by immunofluorescence, co-clusters were counted and their size was measured. To test the role of HA in co-clustering, MDA-MB-231, DT28, and CAF23 cells were treated for 24hrs with 1mM 4-methylumbelliferone (4-MU), then with 2000 U/mL bovine HAase for 1hr. Clustering assays between treated and untreated CTCs and cCAFs were performed as described above with the addition of 1mM 4-MU to the culture. Results: Knock down of CD44 in both MDA-MB-231 and DT28 cells significantly reduced the number and size of co-clusters that were formed with CAF23 cells. In the co-clusters that were formed by CD44-kd 231s and DT28s, CD44 expression returned in these cells while un-clustered 231s and DT28s remained CD44-negative. The removal of HA also significantly reduced the number and size of co-clusters that formed. The reduction in co-clustering caused by HA removal was less than that caused by CD44-kd, suggesting that other ligands or the activity of CD44 is important in co-clustering. Conclusions: These results suggest that co-clustering between CTCs and cCAFs is mediated through CD44 and its ligand, HA. The role of other ligands, such as osteopontin, is currently being tested. The targeting of CD44 and its ligands in breast cancer patients may disrupt co-cluster formation and communication between CTCs and CAFs, reduce the number of circulating CSCs, and prevent the development of metastases. Citation Format: Benjamin Charles Troness, Angela Spartz, Utsav Sharma, James McCarthy, Dorraya El-Ashry. CD44 and its ligand, hyaluronic acid, mediate co-clustering between breast cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5117.

Background: Transport of tumor cells via the circulation (CTCs) is a key step in metastasis. CTC clusters and in particular, cancer stem cell-like cell (cCSC) clusters exhibit enhanced metastatic capacity over individual CTCs or cCSCs. Cancer associated fibroblasts (CAFs) are critical players in the breast tumor microenvironment (TME) and are also found in metastases. In clinical and pre-clinical studies, we have identified circulating CAFs (cCAFs) and cCAF/CTC co-clusters as potential biomarkers for breast cancer progression and metastasis. Our data suggest that the ability of breast cancer cells (BCCs) to cluster with CAFs depends on their intrinsic metastatic capacity, and specifically, their CSC-like phenotype, characterized by expression of the CD44 cell surface marker: both in vivo and in in vitro cluster assays, only metastatic BCCs could form co-clusters while non-metastatic BCCs could not. Further, both in vivo and in vitro, co-clustering potential was marked by a stem cell-like phenotype, including increased CD44 expression. We hypothesize that cCAF/cCSC co-clusters are the functional units of metastasis. The goal of this current study is to identify mechanisms that influence co-clustering between CTCs and cCAFs and specifically to investigate the role of CD44 in co-clustering. Methods: Co- and mono-clusters of CAFs and BCCs were established in vitro by plating equal numbers of CAFs/BCCs into ultra-low attachment plates. After 24 hrs, clusters were collected and injected into female NSG mice via tail vein. Metastases was monitored by IVIS and confirmed by IHC. Mice were sacrificed at specific time points and blood collected by cardiac puncture. In vitro-generated clusters and blood were filtered using the faCTCheker microfluidic filtration instrument (Circulogix) to capture circulating cells with an 8-micron pore filter. Filters were stained by immunofluorescence for tumor cells, CAFs, and CD44. Hyaluronin (HA) was visualized with biotinylated HA binding protein. anti-CD44 siRNA (Dharmacon smart pool) was used to knockdown CD44 in BCCs or in CAFs Results: Injection of CAF/BCC co-clusters resulted in accelerated metastases compared to mono-cluster injection. In both co-clusters captured from mouse blood and established in vitro, HA, a CD44 ligand secreted by CAFs as well as produced by cancer cells, was observed. Elimination of HA in CAFs and BCCs reduced co-cluster formation. Knockdown of CD44 expression in BCCs via siRNA abrogated BCC co-clustering ability. Conclusion: In vitro and in vivo models of induced and conditional knockout of CD44 are defining the role of cancer cell-autonomous or CAF-autonomous CD44 in cCAF/CTC co-clustering and BC metastasis. These studies implicate CD44 as a driver of co-clustering between BCCs and CAFs. Inactivation of CD44 may abrogate co-clustering as well as impair the CSC phenotype, thus impacting critical pathways in BCC metastasis. Citation Format: Benjamin Troness, Angela Spartz, Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Marc Lippman, James McCarthy, Dorraya El-Ashry. CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2044.

Abstract Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Tumor-stromal cell interactions play a pivotal in tumor initiation, progression and metastasis. Cancer-associated fibroblasts (CAFs) are the majority of stroma in BC and are critical to BC tumorigenicity and malignancy. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic BC. cCAFs not only circulate individually, in BC patient blood and in blood from both spontaneous and xenograft murine BC models, but cCAFs are also found in clusters with CTCs. In this study, we examine the egress of CAFs and follow them through to metastatic sites, and evaluate the ability of BC cell subtype/metastatic propensity to influence cCAF and cCAF/CTC cluster egress. Methods: We used NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines, the nonmetastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis to evaluate cCAFs and CTCs in a preclinical model. Mice were sacrificed at specific time points, and cardiac blood was collected to ascertain the temporal dynamics of cCAF and CTC presence. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were enumerated by IF for cCAFs, CTCs, and cCAF/CTC co-clusters. To explore BC-intrinsic factors that influence cCAF and cCAF/CTC cluster egress, we modeled egress from the primary tumor in vitro using transendothelial cell migration assays, where the chemoattractant for CAF cells was conditioned media from the different BC cells lines. Results: In both spontaneous and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. CAFs injected alone in orthotopic xenografts are able to egress independently of BC cells and cCAFs and cCAF clusters were seen; however, co-inoculation with BC cells resulted in substantially higher numbers of both individual cCAFs and cCAF clusters, and now co-clusters were seen. cCAFs appear about 4 days post-injection, and precede appearance of CTCs and CAF/CTC clusters. CAFs co-injected with BC cells into the MFP appear at metastatic sites. In in vitro transendothelial assays, BC secreted factors that potentiate CAF egress are identified as well as the if cCAF/CTC clusters egress as clusters or cluster in the circulation. Conclusion: Although CAFs are highly motile and cCAFs precede CTCs into the circulation, BC subtype influences the ability of CAFs to egress. Targeting cCAF egress and/or cCAF/BC cell clusters provides novel avenues to prevent or treat BC metastasis. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Angela Spartz, Marc Lippman, Dorraya El-Ashry. CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5062.

Abstract Background: Breast cancer metastasis is the cause of breast cancer-related mortality. The tumor microenvironment (TME) plays a critical role in governing tumor initiation, progression and metastasis. In breast cancer, cancer associated fibroblasts (CAFs) are master regulators of the TME. Cancer metastasis occurs in part by transport of circulating tumor cells (CTCs) in the circulation. CTCs in clusters, rather than individual CTCs, have a greater capacity to establish metastases. We recently demonstrated that CAFs also circulate (cCAFs), both in clusters with CTCs and individually, in the blood of the majority of breast cancer patients with metastases, some patients without overt metastases, and in no patients with no evidence of disease. Our lab has also established primary breast cancer and primary CAF cell lines from dissociated breast tumors of different molecular subtypes; these are ideal models to dissect tumor-stromal interactions, both in vitro and in vivo. We hypothesize that cCAFs originate from the primary tumor, and further, that cCAFs cluster with CTCs to facilitate metastatic seeding. Methods: We used separately labeled CAFs and primary breast cancer cells (DT28) co-injected into NSG mice and followed by a novel 2-color IVIS. We also modeled the ability of CAFs and breast cancer cells to form clusters in culture and used our microfluidic filter technology to evaluate the composition of clusters resulting from admixed CAFs and BC cells of differing metastatic capacity – metastatic DT28 and non-metastatic MCF-7. Results: Through sequential dual-color IVIS we demonstrate that we can monitor the presence of CAFs and breast cancer cells at orthotopic tumor injection sites and to sites of distant metastasis, indicating that cCAFs originate from the primary tumor. Evaluation of cCAFs and CTCs in tumor bearing mice indicates that breast cancer cells with high metastatic potential mobilize greater numbers of cCAFs. CAFs formed robust clusters with metastatic DT28 cells, while non-metastatic MCF-7 cells clustered with each other but not with CAFs suggesting that the ability of breast cancer cells to form clusters with CAFs, both in vitro and in vivo, is reflective of the metastatic capability of the breast cancer cell line. Conclusions: The intrinsic metastatic capabilities of breast cancer cells are augmented by contact and clustering with cCAFs. In turn, the ability of CAFs to mobilize into circulation is in part conferred by properties of aggressive breast cancer cells. These data corroborate observations from our pilot clinical study that indicated the presence of cCAFs is overwhelmingly associated with the presence of metastatic breast cancer. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina-Saenz, Pedro Ferrer, Svetlana Speransky, Toni Yeasky, Dorraya El-Ashry. Circulating CAF and cCAF circulating tumor cell co clusters are associated with metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5895. doi:10.1158/1538-7445.AM2017-5895