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.

Chronic stress significantly impacts cancer progression by fostering an immunosuppressive microenvironment that promotes tumor growth and diminishes therapeutic efficacy. Dysregulated stress hormones, like norepinephrine, mediate β-adrenergic stimulation, orchestrating a cascade of immunomodulatory events within the tumor microenvironment (TME). In estrogen receptor-positive (ER+) breast cancer, immunosuppression presents a challenge due to resistance to immunotherapy driven by molecular characteristics dampening antitumor immune responses. We explore the hypothesis that chronic stress, mediated by dysregulated stress hormones, drives immunosuppression in ER+ breast cancer, hindering treatment efficacy. Manipulating thermal stress in the laboratory setting provides a unique approach to studying β-adrenergic stimulation in cancer, particularly its impact on the immune microenvironment. Standard housing conditions for mice result in an environment below their thermoneutral point, inducing increased β-adrenergic stimulation. Conversely, housing mice near their thermoneutral point maintains basal levels of β-adrenergic stimulation, which is associated with delayed tumor growth and improved antitumor phenotypes in different cancer models. Leveraging a syngeneic ER+ breast cancer model, we investigated housing mice at thermoneutral temperatures to mitigate stress-induced immunosuppression. Tumor-bearing mice housed at thermoneutral temperatures exhibited decreased stress hormone levels compared to those housed at standard conditions, correlating with delayed tumor growth. Further analysis revealed notable shifts in immune cell composition within the TME. Mice subjected to thermal stress regulation displayed increased accumulation of CD8+ T cells, reduced PD1 expression on CD8+ T cells, and decreased accumulation of regulatory T cells (Tregs), indicating a shift towards a more immunostimulatory microenvironment. In conclusion, disrupting stress-induced immunosuppression and promoting cytotoxic T cell activity holds profound clinical relevance for individuals with stress-mediated diseases, particularly cancer. By exploring how stress influences immune responses within tumors, our research aims to uncover underlying mechanisms driving tumor progression and immunosuppression in ER+ breast cancer. These insights could inform the development of targeted therapies aimed at modulating stress-related pathways or enhancing immune function to improve treatment outcomes in patients with ER+ breast cancer. Furthermore, understanding the intricate interplay between stress and the immune system may have broader implications for managing stress-related comorbidities in cancer patients, such as anxiety or depression, which can influence immunosuppression, impact treatment efficacy, and overall quality of life. Therefore, the knowledge gained from our study could provide valuable insights into the role of stress in cancer progression and guide the development of therapeutic strategies to mitigate its detrimental effects on the immune response and overall disease outcomes in ER+ breast cancer patients. Citation Format: Gilberto Gastelum Martinez, Marc E. Lippman, Barry Hudson, Philip Miller, Courtney Pine, Yalini Anbalagan. Regulating Stress to Improve Antitumor Immunity in Breast Cancer Models [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-04-19.

Background: The human apolipoprotein E (APOE) gene has three major allelic variants: APOE3, APOE4, and APOE2. Carriers of the APOE4 allele are at high risk for Alzheimer’s and other neurodegenerative disorders and exhibit elevated oxidative stress in the central nervous system. Recently, we observed that mice expressing the human APOE4 allele are predisposed to cardiac damage induced by doxorubicin (DOX), a potent and widely used breast cancer therapeutic. How APOE4 contributes to oxidative stress-related tissue damage remains poorly understood. Objective: To identify mechanisms of APOE4-mediated vulnerability to DOX cardiotoxicity. Methods: C57Bl/6 mice (5-8 mo and 14-18 mo, male and female) with human APOE3 or APOE4 homozygous knock-in (=APOE3 and APOE4, respectively) received a single IP injection of saline (Control) or DOX (10 mg/kg) and monitored between 3-45 days afterward. Cardiac function was quantitated using echocardiography (Vevo 3100) at baseline and during the study. TUNEL assays were used to identify apoptotic cells. Collagen was imaged by Masson’s Trichrome staining. Myocardial protein and RNA were extracted from the left ventricle and subjected to immunoblotting, IHC, real-time PCR, and RNASeq. Transcriptomic data were analyzed using Gene Set Enrichment Analysis (GSEA) for pathway analysis and CIBERSORTx for deconvolution of myocardial immune cell subtypes. Results: At baseline, no difference in cardiac function was observed between the 2 mouse lines. Following DOX treatment, APOE4 mice had greater declines in left ventricular ejection fraction, heart weight, and more myocyte apoptosis compared with age-matched APOE3 mice (all p<0.05). APOE4 also conferred more age-associated myocardial collagen and cardiomyocyte apoptosis than APOE3, and higher myocardial levels of 4-HNE (4-hydroxy-2-nonenal), an oxidative stress byproduct. Surprisingly, APOE4 mice had a marked reduction in immunoglobulin production and age-dependent IgG deposition in the myocardium compared with both APOE3 and wt mice (p= 0.002, E4 vs. E3 at 17 mo). The transcriptomic analysis confirmed defective activation of TGF-beta, TNF-alpha, Myc, and p53 pathway genes in APOE4 mice after DOX, as well as a defective tissue repair response. In APOE3 myocardium, mRNAs encoding neuregulin (Nrg1), a key cardiac survival/repair factor, and multiple cardiac-specific contractile proteins were induced at d3 after DOX; this response was markedly attenuated in APOE4 mice. CIBERSORTx analysis for 22 distinct immune cell types showed an increase in activated M2 macrophages after DOX in APOE3, but not in APOE4 mice. Western and immunohistochemical analyses confirmed increased CD206, a specific M2 macrophage biomarker, in APOE3 but not in APOE4 mice after DOX (p<0.05.) Of note, M2 macrophages increased with age in both wt and APOE3 mice, but not in APOE4 mice (p<0.05, E3 vs. E4, 17 mo.). Conclusion: Our results suggest that the APOE4 allele may confer increased vulnerability to DOX-induced and age-mediated cardiac damage through specific impairment of post-injury repair responses involving M2 macrophage polarization and activation. Whether this is related to known APOE allelic differences in lipoprotein transport or innate immunity functions remains to be determined, as does potential relevance to chemotherapy and other types of cardiac injury in humans. Citation Format: Nanette Bishopric, Harshul Pandit, Adam Ikeda, Kyle Korolowicz, G. William Rebeck, Olga Rodriguez, Marc E. Lippman, Nanette H. Bishopric. APOE4 impairs the cardiac stress response to doxorubicin through defective M2 macrophage activation and tissue repair transcription pathways [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-06-07.

TPS619 Background: Anthracyclines are commonly used to treat high risk early-stage breast cancer (BC). Anthracycline-induced myocardial toxicity is a rare but morbid complication in BC survivors. The mechanism is not completely elucidated but may involve the generation of reactive oxygen species and other inflammatory mediators. RAGE, the receptor for advanced glycation end-products, signaling is upregulated in inflammatory conditions and has been linked to the development and progression of cardiovascular disease and cancer. In experimental models, blocking RAGE protects against cardiac damage and reduces the development of metastases. TTP488 is an orally bioavailable small molecule inhibitor of RAGE. This project is designed to characterize the role of TTP488 to decrease therapy related cardiac toxicity, and it will provide preliminary data about the safety, tolerability, and pharmacokinetics (PK) of common BC (neo)adjuvant chemotherapy agents in the presence of TTP488. Methods: Eligible patients have stage I-III breast cancer and are planned to receive chemotherapy. Enrolled patients will be assigned to 1 of 4 cohorts based on the chemotherapy regimen assigned for the last 2 doses of planned therapy. In Cohort 1, 6 patients will receive dose dense paclitaxel (ddT). In Cohort 2, 6 patients will receive docetaxel and cyclophosphamide (TC). In Cohort 3, 6 patients will receive docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP). In Cohort 4, 6 patients will receive dose dense doxorubicin and cyclophosphamide (ddAC). All chemotherapy is dosed per standard institutional operational practice. For each patient, the first dose of chemotherapy is given in the absence of study drug to serve as the control for the safety and PK assessments. On day -7, prior to the 2nd dose of chemotherapy, TTP 488, 60mg daily for 6 days followed by 20mg daily, is administered and then subjects receive the 2nd dose of chemotherapy. With each cycle, hs-troponin and B-type natriuretic peptide (BNP) are collected prior to chemotherapy administration, and at 4hrs(hs-troponin only) and 24hrs after chemotherapy administration. With each cycle, PK sampling occurs prior to and immediately after chemotherapy (0hr), 1hr, 4hr, and 24hrs after chemotherapy. Primary objectives are to evaluate the change in high sensitivity troponin level before and during treatment with TTP488 and to evaluate the safety and tolerability of TTP488 when administered with chemotherapy. The secondary objective is to characterize the PK and bioavailability of chemotherapy agents with TTP488. Descriptive statistics for the change in troponin level and safety will be summarized. As of 2/1/24, 4 patients have been enrolled, with 4 undergoing screening. At the completion of this trial, we plan a randomized trial to evaluate the role of TTP488 to decrease cardiotoxicity, cancer related cognitive decline and disease recurrence. Clinical trial information: NCT05256745 .

Figure S4. 1,000 HT29 NSC and shRac1b cells were plated in a 96-well plate and treated with different doses of 5FU for 10 days. On day 10, cell viability was analyzed using the PrestoBlue reagent according to the manufacturers instructions. Data was graphed using GraphPad software and IC50 values were generated using the log(inhibitor) vs. response - variable slope function. Data represents quadruplicates and the error bars represent the 95% confidence intervals.

Supplementary Figure 4. Rac inhibition in Prostate Cancer. (A) Migration Assay on DU145 cells treated with vehicle or GYS32661. (B) Pan-Kinase RPPA analysis of LnCaP cells treated with vehicle or GYS32661.

Supplemental Figure 1. Rac1 is a driver of ccRCC. (A) Rac1 mRNA expression in normal kidney and ccRCC patients from Jones Renal Cohort. (B) Rac1 DNA copy number analysis of normal blood, normal kidney, and kidney tumors from the KIRC TCGA dataset. (C) Rac isoform expression analysis in normal kidney and kidney tumors from the TCGA KIRC dataset. (D) PREX1, VAV1, TIAM1, and TRIO mRNA expression in normal kidney and kidney tumors from the TCGA KIRC dataset. (E) Rac1 mRNA expression by clinical stage from the TCGA KIRC dataset. (F) Boyden chamber cellular migration assay of 786-O transfected with siNSC or siRac1 oligonucleotides. (G) Cellular proliferation assay of 786-O transfected with siNSC or siRac1 oligonucleotides.

Figure S7. HCT116 cells were treated with Oxaliplatin (5 μM) and GYS32661 (25 μM) and the combination of two, stimulated with TNFα for 10 mins. 50 μg of protein was resolved on SDS-PAGE gel, transferred on the nitrocellulose member. The membrane was probed with phosho IκÎ'α, total IκÎ'α, and phospho P65 antibodies

Figure S5. HCT116 cells were treated with different dose of 5FU (6.25, 12.5, 25 μM) for 72hrs. The cells were lysed in RIPA lysis buffer. 50 μg of protein was resolved on SDS-PAGE gel, transferred on the nitrocellulose member. The membrane was probed with Rac1b, Rac1 and actin antibodies.

Table S1 Shows Rac1 Immunohistochemistry scores for normal colon and colon cancer by stage

Figure S1 shows Rac1 expression analysis of Normal Colon and Colon Adenocarcinoma patients obtained from the TCGA COAD dataset using Oncomine. Data is represented as Log2 Median centered ratio.

Supplementary Figure 2. Cellular proliferation assay of VCaP cells expressing either control shRNA (shNSC) or two independent HACE1 shRNAs (shHACE1 1 and shHACE1 2).

Figure S6. (A) Five different colorectal cancer cells showing the sensitivity for GYS32661, KRAS and BRAF mutation and Rac1b expression. (B) HCT116-EV and HCT116-Rac1b cells stably expressing the NFκÎ' reporter was treated with different dose of GYS32661. The cells were lysed in reporter lysis buffer and NFκB reporter activity was measured. The results, presented as the NF-kB-mediated luciferase activity normalized to the protein concentration are the means {plus minus} SEM from three independent experiments. (C) Mice body weight following treatment with GYS32661.

Supplemental Figure 2. HACE1 overexpression reduces activated Rac1 and attenuates ccRCC growth and survival. (A) Rac1 and HACE1 mRNA expression correlation from the KIRC TCGA Dataset (B) Rac1-GTP Pull-down assay of empty vector or HACE1 overexpressing 786-O cells. (C) Migration assay on empty vector or HACE1 overexpressing 786-O cells. (D) Cellular proliferation assay of empty vector or HACE1 overexpressing 786-O cells. (E) Soft agar assay results of empty vector or HACE1 overexpressing 786-O cells.

Supplemental Figure 3. In vitro and In Vivo characterization of GYS32661. (A) Cellular proliferation assay of 786-O cells treated with vehicle, 2.5, and 5 μM GYS32661. (B) Plasma and tumor concentrations of GYS32661 at 0.25, 1, 4, and 8 hour time points.

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.

Figure S2. (A) Chemical structure of JSH-23. (B) HCT116-EV and HCT116-Rac1b cells stably expressing the NFκÎ' reporter was treated with different dose of JSH-23 for 4 hours. The cells were lysed in reporter lysis buffer and NFκB reporter activity was measured.