Preclinical Models Of Breast Cancer Research Articles

Abstract SP090: Tumor INF signaling - A double edged sword in cancer

Abstract Our pre-clinical research using breast cancer models has revealed how certain inflammatory pathways can have opposing effects on primary disease and metastasis. We also learned that immunological age can impact response to therapy, leading to age-stratified outcomes. In one study, we developed pre-clinical models of triple-negative breast cancer (TNBC) lung metastasis. We uncovered mechanisms by which certain primary breast cancers can prevent their disseminated tumor cells from generating tumors. These primary tumors elicit a systemic innate immune response involving IL-1β-expressing neutrophils that infiltrate the lungs. Our group and others had previously established that IL-1β promotes primary breast cancer growth. However, we found that in the lungs, IL-1β maintains disseminated tumor cells in an undifferentiated, non-proliferative state. Inhibiting either inflammation or IL-1R signaling enables robust outgrowth of lung metastases. Importantly, we found that among patients with lymph node-positive breast cancer (i.e., those at high risk for metastasis) high IL-1β associates with improved overall survival and distant metastasis-free survival. Our findings reveal a double-edged sword in which IL-1R inhibitors might reduce primary disease but promote metastatic disease. To avoid this dichotomy, we are exploring strategies that prevent disseminated tumor cell differentiation, as was achieved here by IL-1β, to prevent metastatic outgrowth and provide a window of opportunity to target early metastases. As another example, we evaluated immune checkpoint blockade (ICB) therapy using various pre-clinical TNBC models in young and aged mice. We observed reduced tumor growth and improved survival in response to anti-PD-1 and anti-CTLA-4 treatment in young mice compared to aged mice. Immune profiling of peripheral blood and tumor samples demonstrated differences between young and aged mice treated with immune checkpoint blockade. To gain insights into the mechanisms behind those results, we performed whole tumor RNAseq on treated young versus aged mice. Notably, the most significantly enriched gene signatures included genes in interferon response pathways - with impaired responses in tumors of aged mice. Informed by those findings, we interrogated the METABRIC tumor gene expression database, stratifying TNBC patients < 40 and > 65 years of age. Two distinct clusters were identified, including enrichment of interferon-related genes in the "young" cohort. Turning back to our pre-clinical models, we used a drug that stimulates interferon response genes (STING agonists) in combination with ICB and found significantly improved survival in the aged cohorts. Our study demonstrated that age determines the T cell inflamed phenotype and affects response to immune checkpoint blockade in TNBC. Evaluating interferon-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an interferon pathway activator with ICB may be required. These findings highlight the need to understand how inflammatory signatures should be interpreted in order to design more effective clinical phase trials and how pre-clinical models need to better mimic intent-to-treat populations. Citation Format: SS McAllister. Tumor INF signaling - A double edged sword in cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP090.

Abstract PS17-49: Investigating CDK4/6 inhibition in triple negative breast cancer

Abstract The purpose of our study is to evaluate the immunological effects of CDK4/6 inhibitors in pre-clinical models of triple negative breast cancer (TNBC). CDK4/6 inhibitors, including abemaciclib, are currently approved to treat patients with metastatic hormone receptor-positive (HR+), Her2-negative breast cancer. We previously reported in our Her2+ model that CDK4/6 inhibitors promote anti-tumor immunity through inducing tumor cell antigen presentation via activating tumor endogenous retroviral elements, which induce type III interferon production and MHC-I upregulation (Goel, DeCristo, et al., Nature, 2017). We also uncovered important anti-tumor effects on the immune system: CDK4/6 inhibitors decrease T regulatory cell proliferation without affecting cytotoxic CD8 T cell numbers. TNBC has been considered a poor candidate for CDK4/6 inhibitor therapy, as tumors often lose retinoblastoma (Rb) protein expression/function, which is critical for CDK4/6 inhibitor-induced cell cycle arrest. However, Rb mutation or loss occurs in only ~20% of TNBC cases and we found Rb expressing murine and human TNBC cell lines decreased proliferation in vitro in response to abemaciclib. In our preclinical model of TNBC, abemaciclib induced tumor regression. Consistent with previous findings, TNBC tumor cells upregulated MHC-I upon abemaciclib treatment, suggesting increased antigen presentation. Tumor cell-surface PD-L1 was also increased with abemaciclib both in vitro and in vivo, as assessed by flow cytometry and RT-qPCR. These results are encouraging, given that αPD-L1 therapy (Atezolizumab) in combination with chemotherapy (nab-paclitaxel) has recently been approved as standard care for metastatic TNBC and the IMpassion130 trial reported enhanced progression-free and overall survival in patients with PD-L1+ tumors. We also found that abemaciclib increased CD8+ and CD4+ T cells and decreased PD1+ CD8 and CD4 cells in the spleen. Furthermore, total numbers of naïve CD8+ and CD4+ T cells increased with abemaciclib treatment, suggesting a favorable anti-tumor systemic immunological effect. Our data suggest the potential efficacy of CDK4/6 inhibitors in combination with αPD-L1 in the treatment of Rb+ TNBC. Deeper analysis of the mechanisms involved in regulating PD-L1 and enhancing naïve T cells should enable us to evaluate combination therapies using CDK4/6 inhibitors for this particularly deadly breast cancer subtype. Citation Format: Qiuchen Guo, Gregory J Goreczny, Milos Spasic, Adam Maynard, Sandra S McAllister. Investigating CDK4/6 inhibition in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-49.

Abstract PS10-30: An open label, pilot study of veliparib and lapatinib in patients with metastatic, triple negative breast cancer

Abstract Purpose: Poly (ADP-ribose)-polymerase inhibitors (PARPi) have been approved for cancer patients with germline BRCA1/2(gBRCA1/2) mutations, and efforts to expand the utility of PARPi beyond BRCA1/2 are ongoing. In preclinical models of triple negative breast cancer (TNBC) with intact DNA repair, we previously showed an induced synthetic lethality with combined EGFR inhibition and PARPi. We report the safety and clinical activity of lapatinib and veliparib in patients with metastatic TNBC. Experimental Design: A first-in-human, pilot study of lapatinib and veliparib was conducted in metastatic TNBC (NCT02158507). The primary endpoint was safety and tolerability. Secondary endpoints were objective response rates and pharmacokinetic evaluation. Gene expression analysis of pre-treatment tumor biopsies was performed. Key eligibility included TNBC patients with measurable disease and prior anthracycline and taxane therapy. Patients with gBRCA1/2 mutations were excluded. Results: Twenty patients were enrolled of which 17 were evaluable for response. Median number of prior therapies in the metastatic setting was 1 (range 0-2). Fifty percent of patients were Caucasian, 45% African-American, and 5% Hispanic. Of evaluable patients, 4 demonstrated a partial response and 2 had stable disease. There were no dose-limiting toxicities. Most AEs were limited to grade 1 or 2 and no drug-drug interactions noted. Gene expression analysis suggest baseline DNA repair pathway score was lower and baseline immunogenicity was higher in the responders compared to non-responders. Conclusions: Lapatinib plus veliparib therapy has a manageable safety profile and promising antitumor activity in advanced TNBC. Further investigation of dual therapy with EGFR inhibition and PARP inhibition is needed. Citation Format: Erica M Stringer-Reasor, Jori E May, Eva Olariu, Valeria Caterinicchia, Yufeng Li, Deborah Della Manna, Gabrielle B Rocque, Christos Vaklavas, Carla I Falkson, Lisle M Nabell, Edward P Acosta, Andres Forero-Torres, Eddy S Yang. An open label, pilot study of veliparib and lapatinib in patients with metastatic, triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-30.

Abstract PS3-28: Three-dimensional H-scan ultrasound imaging for acute detection of breast cancer response to neoadjuvant treatment - Initial results using a preclinical animal model

Abstract Introduction: The use of noninvasive ultrasound (US) for tumor tissue characterization is an exciting prospect for anticancer treatment response monitoring. To that end, our group has developed a new technology termed H-scan US imaging, which links differences in the raw backscattered US signals to various-sized tissue structures. The purpose of this research project was to develop a 3-dimensional (3D) H-scan US imaging system and method for tissue characterization in volume space and evaluate using a preclinical animal model of breast cancer. Methodology: Preliminary studies were conducted using female nude athymic mice (N = 20, Charles River Laboratories) implanted in the mammary fat pad with 1 million breast cancer cells (MDA-MB-231, ATCC). Once tumors reached about 1 cm in size, animals were sorted into four groups so that mean tumor size in each was comparable (N = 5 per group). Animals were then US imaged at baseline and before receiving intraperitoneal injections, namely: (1) 0.3 mg sterile saline (control), (2) 0.2 mg of agnostic TRA-8 monoclonal antibody to human death receptor 5 (DR5) + 0.1 mg sterile saline, (3) 0.1 mg paclitaxel + 0.2 mg sterile saline, and (4) 0.1 mg TRA-8 + 0.2 mg paclitaxel. Image data was acquired using a programmable US scanner (Vantage 256, Verasonics Inc) equipped with a volumetric imaging transducer (4DL7, Vermon) at baseline and again every 24 hours for 3 days. To generate the H-scan US images, a set of Gaussian-weighted Hermite filters were convolved with the radiofrequency (RF) data to measure the relative strength of the received signals. The signal envelope for each of the filtered signal then was calculated using a Hilbert transformation. Finally, the lower frequency backscattered signals were assigned to a red (R) channel and the higher frequency components to a blue (B) channel. The unfiltered original RF signal was assigned to the green (G) channel to complete the RGB colormap and 3D H-scan US image display. After US imaging on day 3, animals were humanely euthanized and tumors excised for histological processing using the TUNEL HRP-DAB assay and immunofluorescent staining for activated caspase-3, PARP1, Ki-67, and DAPI. Results: The in vivo results show that 3D H-scan US imaging is considerably more sensitive to tumor changes after neoadjuvant treatment as compared to traditional B-scan US. While there was no difference at baseline (p = 0.52), repeat H-scan US results from treated tumors exhibited progressive increases in image intensity (164.9 ± 23.4%, 180.29 ± 11.6%, and 229.2 ± 14.6% for groups 2, 3, and 4 at day 3, respectively; p < 0.05) due to cancer cell nuclear condensation and apoptotic activity. Moreover, 3D H-scan US exhibited less variance than planar measurements due to increased sample size and statistical averaging. Histological findings also confirmed increased apoptosis and decreased proliferation that matched H-scan US data trends. Conclusions: 3D H-scan US imaging is a promising technique that allows visualization of the heterogenous tissue microenvironment and improves the evaluation of treatment at an early stage of therapy as validated by histologic findings. Citation Format: Haowei Tai, Shreya Reddy, Jane Song, Mawia Khairalseed, Kenneeth Hoyt. Three-dimensional H-scan ultrasound imaging for acute detection of breast cancer response to neoadjuvant treatment - Initial results using a preclinical animal model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-28.

Abstract OT-09-05: A randomized, pre-surgical study to investigate the biological effects of AZD9833 doses in women with ER-positive HER2-negative primary breast cancer (SERENA-3)

Abstract Background AZD9833 is an orally bioavailable selective estrogen receptor (ER) antagonist and degrader (SERD) that has shown anti-tumor efficacy in a range of preclinical breast cancer models. SERENA-1, an ongoing first-in-human study assessing AZD9833 as a monotherapy and in combination with palbociclib, established dose-dependent tolerability in pre- and post-menopausal women at doses of 25-450 mg once daily (QD), with clinical benefit and target engagement at all dose levels. Two randomized, open-label Phase 2 trials are also ongoing in women with ER+ HER2- breast cancer. SERENA-2 compares the efficacy of AZD9833 with fulvestrant in post-menopausal women with advanced breast cancer following treatment with ≤1 endocrine therapy. SERENA-3 will examine the biological effects of different doses of AZD9833 in treatment-naïve women with primary breast cancer. Methods SERENA-3 is a randomized, open-label, parallel-group, pre-surgical study to investigate the biological effects of different doses of AZD9833 in ER+, HER2- primary breast cancer. Eligible patients will be post-menopausal (and potentially pre-menopausal) women awaiting curative-intent surgery for newly diagnosed, ER+ HER2- primary breast cancer. The study is designed in two stages. In Stage 1, 24 post-menopausal women will be randomized 1:1 to receive either 75 mg or 150 mg oral AZD9833 QD for 5-7 days, followed by a minimum 5-day pre-surgery washout period; Stage 2 gives provision for additional groups depending on emerging data from Stage 1. The primary objective of this study is to explore the effect of AZD9833 on ER expression in pre- and on-treatment tumor samples from women with primary breast cancer, as assessed by immunohistochemistry and H-score. Safety and tolerability will be assessed as secondary endpoints, along with the pharmacokinetic and pharmacodynamic effects of AZD9833 on other biomarkers. Blood will be collected at screening, on the day of biopsy and the day of surgery to assess circulating tumor DNA and exploratory biomarkers. Primary endpoint analysis will be performed on the pharmacodynamic analysis set. The study will be conducted in 20 centers across 3 countries. For more information please contact Professor John Robertson at: john.robertson@nottingham.ac.uk. Citation Format: John FR Robertson, Iain Moppett, Juan Enrique Bargalló Rocha, Giorgi Dzagnidze, Joanna Harding, Teresa Klinowska, Richard Mather, Alastair Mathewson, Rhiannon Maudsley, Christopher J Morrow, Andrew Saunders, Andy Sykes, Li Zhang, Justin PO Lindemann. A randomized, pre-surgical study to investigate the biological effects of AZD9833 doses in women with ER-positive HER2-negative primary breast cancer (SERENA-3) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-05.

Abstract PR011: Selective inhibition of VEGF binding to VEGFR2 promotes an immune stimulatory microenvironment in murine models of breast cancer

Abstract Vascular endothelial growth factor-A (VEGF) is the dominant angiogenic factor that promotes tumor neovascularization, making it a critical target for cancer therapy. VEGF binds and activates VEGF receptor 1 (VEGFR1) and VEGFR2, with VEGFR2 being the principal receptor mediating VEGF-induced angiogenesis while VEGFR1 has a negative regulatory effect on this process. Previously, we found that selective inhibition of VEGF binding to VEGFR2 with a fully human monoclonal antibody (r84) or its mouse chimeric counterpart (mcr84) is sufficient for effective control of tumor growth in preclinical models of breast cancer. However, accumulating evidence indicates that the effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. Concordantly, we have shown that VEGF blockade regulates immune cell trafficking across the endothelium by increasing the expression of adhesion molecules while decreasing FasL expression on tumor vasculature. In addition, specifically blocking VEGF binding to VEGFR2 promotes an immune stimulatory microenvironment by enhancing activation of CD8+ cytotoxic T cells as evidenced by elevated secretion of Granzyme B and IFN-γ. In addition, VEGF blockade also reduces FoxP3+ regulatory T cell infiltration in multiple murine models of cancer. We and others have shown that immune cells express receptors for VEGF. Our data indicate that VEGFR2 expression is elevated on myeloid cells selectively in tumor-bearing animals. Consistent with VEGFR2 expression, treatment with mcr84 results in a reduction in macrophage/myeloid cell infiltration into murine breast tumors. In conclusion, our results enhance understanding of the function of VEGFR2 signaling on myeloid cells and suggest that selective inhibition of VEGF from binding VEGFR2 facilitates immune activation landscape in breast cancer models, which might enhance the efficacy of immune checkpoint blockade. This abstract is also being presented as PO045. Citation Format: Yuqing Zhang, Huocong Huang, Noah Sorrelle, Rolf Brekken. Selective inhibition of VEGF binding to VEGFR2 promotes an immune stimulatory microenvironment in murine models of breast cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR011.

Effects of HER Family–targeting Tyrosine Kinase Inhibitors on Antibody-dependent Cell-mediated Cytotoxicity in HER2-expressing Breast Cancer

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array-determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry-based protocols. Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell-mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. TKIs differentially alter tumor cell phenotype which can impact NK cell-mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Targeting transcription of MCL-1 sensitizes HER2-amplified breast cancers to HER2 inhibitors

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.

Stromal Platelet-Derived Growth Factor Receptor-β Signaling Promotes Breast Cancer Metastasis in the Brain.

Platelet-derived growth factor receptor-beta (PDGFRβ) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that mediates both primary tumor growth and metastasis, specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells, while mesenchymal-specific expression of an activating mutant PDGFRβ (PDGFRβD849V) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRβD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRβD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRβD849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRβD849V in their stromal cells as a preclinical model of breast cancer-associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRβ signaling in women with breast cancer. SIGNIFICANCE: These studies reveal a previously unknown role for PDGFB-to-PDGFRβ paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients.See related article by Wyss and colleagues, p. 594.

Deregulated Immune Pathway Associated with Palbociclib Resistance in Preclinical Breast Cancer Models: Integrative Genomics and Transcriptomics.

Recently, cyclin-dependent kinase (CDK) 4/6 inhibitors have been widely used to treat advanced hormone receptor-positive breast cancer. Despite promising clinical outcomes, almost all patients eventually acquire resistance to CDK4/6 inhibitors. Here, we screened genes associated with palbociclib resistance through genomics and transcriptomics in preclinical breast cancer models. Palbociclib-resistant cells were generated by exposing hormone receptor-positive breast cancer cell lines to palbociclib. Whole-exome sequencing (WES) and a mRNA microarray were performed to compare the genomic and transcriptomic landscape between both palbociclib-sensitive and resistant cells. Microarray analysis revealed 651 differentially expressed genes (DEGs), while WES revealed 107 clinically significant mutated genes. Furthermore, pathway analysis of both DEGs and mutated genes revealed immune pathway deregulation in palbociclib-resistant cells. Notably, DEG annotation revealed activation of type I interferon pathway, activation of immune checkpoint inhibitory pathway, and suppression of immune checkpoint stimulatory pathway in palbociclib-resistant cells. Moreover, mutations in NCOR1, MUC4, and MUC16 genes found in palbociclib-resistant cells were annotated to be related to the immune pathway. In conclusion, our genomics and transcriptomics analysis using preclinical model, revealed that deregulated immune pathway is an additional mechanism of CDK4/6 inhibitor resistance besides the activation of cyclin E-CDK2 pathway and loss of RB, etc. Further studies are warranted to evaluate whether immune pathways may be a therapeutic target to overcome CDK4/6 inhibitor resistance.

Gut Microbiota Condition the Therapeutic Efficacy of Trastuzumab in HER2-Positive Breast Cancer.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B-positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. SIGNIFICANCE: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients.See related commentary by Sharma, p. 1937 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2195/F1.large.jpg.

Loss of type I IFN responsiveness impairs natural killer cell antitumor activity in breast cancer.

Competent type I IFN signaling is the lynchpin of most immune surveillance mechanisms and has recently proven critical to the efficacy of several anticancer agents. Expression of the type I IFN receptor, IFNAR, underpins type I IFN responsiveness in all cells and facilitates the activation and cytotoxic potential of lymphocytes, while loss of IFNAR on lymphocytes has previously been associated with tumor progression and poor patient survival. This study underscores the importance of intact type I IFN signaling to NK cells in the regulation of tumorigenesis and metastasis, whereby ablation of NK cell IFNAR1 impairs antitumor activity and tumor clearance. Using a preclinical model of triple negative breast cancer, we identified that intact IFNAR on NK cells is required for an effective response to type I IFN-inducing immunotherapeutics that may be mediated by pathways associated with NK cell degranulation. Taken together, these data provide a rationale for considering the IFNAR status on NK cells when devising therapeutic strategies aimed at inducing systemic type I IFN signaling in breast cancer.

Short-term CDK4/6 Inhibition Radiosensitizes Estrogen Receptor-Positive Breast Cancers.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor-positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models. Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy. Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21-1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08-1.68) and abemaciclib-treated (rER: 1.19-2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94-1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth. These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

RX-5902, a novel \u03b2-catenin modulator, potentiates the efficacy of immune checkpoint inhibitors in preclinical models of triple-negative breast Cancer

BackgroundTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear β-catenin signaling. The purpose of this study was to evaluate the ability of β-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC.MethodsTreatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2nullIl2rγnullSIRPαNOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells.ResultsThe addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p < 0.05). In the RX-5902/nivolumab combination group, there was a significant increase in the percentage of CD4+ T cells in TILs and increased systemic granzyme B production (p < 0.01).ConclusionsConclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

Preclinical Models to Study Obesity and Breast Cancer in Females: Considerations, Caveats, and Tools.

Obesity increases the risk for breast cancer and is associated with poor outcomes for cancer patients. A variety of rodent models have been used to investigate these relationships; however, key differences in experimental approaches, as well as unique aspects of rodent physiology lead to variability in how these valuable models are implemented. We combine expertise in the development and implementation of preclinical models of obesity and breast cancer to disseminate effective practices for studies that integrate these fields. In this review, we share, based on our experience, key considerations for model selection, highlighting important technical nuances and tips for use of preclinical models in studies that integrate obesity with breast cancer risk and progression. We describe relevant mouse and rat paradigms, specifically highlighting differences in breast tumor subtypes, estrogen production, and strategies to manipulate hormone levels. We also outline options for diet composition and housing environments to promote obesity in female rodents. While we have applied our experience to understanding obesity-associated breast cancer, the experimental variables we incorporate have relevance to multiple fields that investigate women's health.

Data-Driven identification of chemopreventive agents for breast cancer

Preclinical animal models of breast cancer provide the opportunity to identify chemopreventive drugs with single-agent activity as well as effective multi-modality regimens for primary as well as secondary prevention in high-risk persons. Our group has used the 7,12-dimethylbenz(a)anthracene (DMBA) mouse model of carcinogen-induced breast cancer to explore the clinical potential of two tyrosine kinase inhibitors and a nucleoside analog as chemopreventive agents. All three agents exhibited promising preclinical activity both as monotherapy and as components of combination therapy with the standard chemotherapy drug paclitaxel. The tumors developing despite chemoprevention were not only small and grew slowly, but they also displayed a uniquely more pro-apoptotic protein expression profile. Hence, our experimental chemopreventive drugs were capable of preventing the development of aggressive mammary gland tumors with an apoptosis-resistant protein expression profile.

Therapeutic potential of Fingolimod in triple negative breast cancer preclinical models

Surgery followed by a chemotherapy agent is the first-line treatment for breast cancer patients. Nevertheless, new targets are required for women with triple-negative breast cancer (TNBC) in order to improve the treatment of this aggressive cancer subtype. Multiple pro-inflammatory molecules including lipid-based substances such as sphingosine-1-phosphate (S1P) promote cancer progression. In this preclinical study, we aim to investigate the efficacy of Fingolimod, an inhibitor of S1P / S1P receptors axis, already approved as an immunomodulator in multiple sclerosis.The impact of Fingolimod was analyzed using in vitro 2D and 3D cell survival analysis and in vivo orthotopic graft models, using mouse and human TNBC cells implanted in immunocompetent or immunodeficient mice, respectively. Resection of the tumor primary mass was also performed to mimic the clinical standard of care.We demonstrated that Fingolimod repressed tumor cell survival in vitro. We also showed in preclinical mouse TNBC models that Fingolimod repressed tumor progression and liver and spleen metastases without apparent adverse effects on the animals. Our data indicate that Fingolimod induces tumor cells apoptosis and thereby represses tumor progression.Globally, our data suggest that Fingolimod merits further evaluation as a potential therapeutic opportunity for TNBC.

Preclinical Development of MGC018, a Duocarmycin-based Antibody–drug Conjugate Targeting B7-H3 for Solid Cancer

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.

3,3'-Diindolylmethane Enhances Tumor Regression After Radiation Through Protecting Normal Cells to Modulate Antitumor Immunity

PurposePreclinical and clinical data indicate that radiation therapy acts as an immune modifier, having both immune-stimulatory and immunosuppressive effects on the tumor-immune microenvironment (TIME). 3.3’-diindolylmethane (DIM) sensitizes tumor cells to radiation and protects mice from lethal doses of total body irradiation. We hypothesize that protecting nontumoral cells from the adverse effects of radiation treatment (RT) may help to correct immunosuppression resulting from radiation. Methods and MaterialsWe generated tumor graft models using immune-competent and immune-deficient mouse strains. Narrow-beamed radiation was targeted to tumor sites using shielding. Tumor regression was monitored after DIM and RT versus RT alone. The effects of DIM on the efficacy of RT were assessed using immunohistochemistry staining and gene expression profiling. Complete blood counts, clonogenic cell survival assays, and global gene expression profiling of cultured cells were performed to study DIM’s radioprotective effects on normal cells. ResultsDIM enhanced tumor regression after RT in immune-competent but not immune-deficient mice. Data indicated that DIM increased intratumoral immune cells after RT, contributing to enhanced immunologic responses such as adhesion and antigen processing. DIM protected normal cells from radiation-induced immediate injuries in vitro and in vivo. Transcriptomic profiling of cultured cells showed that DIM treatment mildly increased expression of some genes that are normally induced after radiation, such as genes involved in cell cycle arrest and apoptosis. ConclusionsIn this study, using cultured cells and preclinical breast cancer models, we show that DIM protects normal cells from radiation-induced immediate cellular injury and combination treatment of DIM and radiation potentiates antitumor immune responses and enhances the efficacy of RT.