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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.

The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence. Significance: Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. Cancer Res; 78(18); 5300–14. ©2018 AACR.

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 HR+ model that CDK4/6 inhibitors promote anti-tumor immunity by 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 suppressed tumor growth 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. However, treatment with Palbociclib did not increase tumor PD-L1 level, suggesting the potential functional variances between different CDK4/6 inhibitors. Our data therefore suggest the potential efficacy of abemaciclib in combination with αPD-L1 in the treatment of Rb+ TNBC. 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. We recently performed RNAseq on abemaciclib-treated TNBC and while we do not have the results at this time, they will be analyzed and prepared for presentation at the meeting. Deeper analysis of the mechanisms involved in regulation of PD-L1 expression and enhancement of 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, Milos Spasic, Gregory Goreczny, Adam Maynard, Sandra McAllister. Investigating anti-tumor immunity with CDK4/6 inhibition in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1955.

Breast cancer affects women of all ages yet young (65) experience worse survival than their middle-aged counterparts. Age is associated with increasing immune dysfunction, yet the clinical implications of age-related changes are unknown, as women 65 years of age are under-represented in clinical trials. We hypothesized that both young and older immunological age impact breast cancer outcomes and present unique obstacles to therapeutic efficacy. Using pre-clinical triple-negative breast cancer (TNBC) models, we had found that bone marrow progenitor cells upregulate granulin (GRN), a secreted pro-inflammatory factor. Once mobilized from the marrow, those cells infiltrate tumors where GRN generates a tumor-supportive microenvironment. High GRN also correlated with reduced survival in our cohort of breast cancer patients. When we performed the same experiments in immunologically old mice (12-18 mos), tumors grew slower and GRN+ cell recruitment was reduced. GRN+ bone marrow cells from young donor mice rescued tumor growth in aged recipients, indicating that pro-tumor immunity resides in the marrow and explaining the aggressive TNBC growth in young cohorts. We next asked if age impacts immune checkpoint blockade (ICB) efficacy, using a 4T1 TNBC model to evaluate anti-PD-L1 or anti-CTLA4 monotherapy. Young mice responded to anti-PD-L1 and anti-CTLA4 with significantly prolonged survival; however, aged mice derived no benefit from ICB therapy. In response to ICB, tumors in young mice had significantly higher CD8+:Treg ratios and were enriched for inflammatory and interferon (IFN)-gamma response genes, thus revealing active anti-tumor immunity in young but not aged mice. Importantly, IFN-alpha, IFN-gamma, and inflammatory pathways were enriched in young relative to aged isotype control mice. When we analyzed METABRIC gene expression data for TNBC patients 65, IFNg, IFNa, and inflammatory responses were enriched only in patients 65. To stimulate immune priming in aged mice, we evaluated the STING agonist, DMXAA, which significantly prolonged survival in combination with ICB in the aged cohort. We are currently performing a pre-clinical study mirroring the IMpassion031 trial, which recently reported improved pathological complete response rates to neoadjuvant atezolizumab (anti-PD-L1)+nab-paclitaxel in TNBC. In a Met1 TNBC model, tumors grew slower with improved survival in the untreated aged mice. Young and aged mice responded to paclitaxel alone and anti-PD-L1 monotherapy; however, anti-PD-L1 provided additional benefit to paclitaxel only in young mice, similar to the trend reported in the IMpassion031 trial. Our results support a model whereby the net effects of pro- and anti-tumor immunity change with age and affect breast cancer outcomes. Understanding age-related immune function should enable us to design age-stratified breast cancer therapies. Citation Format: Sandra S. McAllister. TIME and Age: Impact of age on the tumor immune microenvironment and response to therapy [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 IA023.

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.

Triple-negative breast cancer (TNBC) is considered an early onset subtype of breast cancer that carries with it a poorer prognosis in young rather than older women for reasons that remain poorly understood. Hematopoiesis in the bone marrow becomes altered with age and may therefore affect the composition of tumor-infiltrating hematopoietic cells and subsequent tumor progression. In this study, we investigated how age- and tumor-dependent changes to bone marrow–derived hematopoietic cells impact TNBC progression. Using multiple mouse models of TNBC tumorigenesis and metastasis, we found that a specific population of bone marrow cells (BMC) upregulated CSF-1R and secreted the growth factor granulin to support stromal activation and robust tumor growth in young mice. However, the same cell population in old mice expressed low levels of CSF1R and granulin and failed to promote tumor outgrowth, suggesting that age influences the tumorigenic capacity of BMCs in response to tumor-associated signals. Importantly, BMCs from young mice were sufficient to activate a tumor-supportive microenvironment and induce tumor progression in old mice. These results indicate that hematopoietic age is an important determinant of TNBC aggressiveness and provide rationale for investigating age-stratified therapies designed to prevent the protumorigenic effects of activated BMCs. Cancer Res; 76(10); 2932–43. ©2016 AACR.

Immune checkpoint blockade therapy has revolutionized the treatment of many cancer types and has proven effective for ~10-20% of breast cancer patients in early clinical trials. For patients with triple-negative breast cancer, these responses represent an improvement over standard therapeutic strategies, offering hope that improving efficacy of immunotherapies will save patient lives. However, the reasons for resistance or lack of response to these therapies in breast cancer are unknown. First, we made preclinical observations that provide rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anticancer treatment. Using various preclinical breast cancer models, we showed that selective CDK4/6 inhibitors cause tumor regression by promoting antitumor immune responses. Genome-wide expression data from patient tumors in the NeoPalAna trial revealed similar antitumor immune signatures. The antitumor immunity occurs through processes that affect both tumor cells and the immune microenvironment. Hence, disease regression was further enhanced using a combination therapy regimen comprising a CDK4/6 inhibitor with immune checkpoint blockade. Second, we found that age-related changes to immune function present limitations to the application of immunotherapy in our triple-negative breast cancer (TNBC) preclinical models. While TNBC incidence is similar in young and old patients, it is not known whether age impacts response to immunotherapy in breast cancer. In fact, while over 50% of patients are over 65 years of age at diagnosis, elderly individuals are often excluded from clinical trials for various reasons. We found that aged mice (>12 months, corresponding to ~65-year old humans) had significantly reduced ability to respond to either anti-PD-L1 or anti-CTLA4 compared to young counterparts (8-10 weeks, corresponding to ~25-year old humans). Using high-content flow cytometric analyses and RNA-seq, we identified cellular and molecular mechanisms that correlate with response in young mice that are attenuated in old mice. Many of the differentially expressed genes that defined response in the young cohorts and resistance in the older cohorts were also predictors of response to immunotherapy in cancer patients. Our data suggested novel combination therapies that, when combined with checkpoint blockade, improved responses and survival in the old TNBC cohort. By studying pharmacologic and physiologic processes that improve responses, we hope to suggest new treatment strategies that ensure that breast cancer patients see the most benefit possible from checkpoint blockade therapy. Citation Format: Sandra S. McAllister. Pharmacologic and physiologic processes that affect response to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA22.

Immune checkpoint blockade (ICB) therapy, which targets T cell–inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple-negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor-infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10% to 15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and patients with TNBC shows decreased IFN signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in patients with TNBC receiving ICB therapy. Significance: These data demonstrate for the first time that age determines the T cell–inflamed phenotype in TNBC and affects response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required. This article is highlighted in the In This Issue feature, p. 1143

Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1β (IL-1β)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1β maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1β expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.

The causes for malignant progression of disseminated tumors and the reasons recurrence rates differ in women with different breast cancer subtypes are unknown. Here, we report novel mechanisms of tumor plasticity that are mandated by microenvironmental factors and show that recurrence rates are not strictly due to cell-intrinsic properties. Specifically, outgrowth of the same population of incipient tumors is accelerated in mice with triple-negative breast cancer (TNBC) relative to those with luminal breast cancer. Systemic signals provided by overt TNBCs cause the formation of a tumor-supportive microenvironment enriched for EGF and insulin-like growth factor-I (IGF-I) at distant indolent tumor sites. Bioavailability of EGF and IGF-I enhances the expression of transcription factors associated with pluripotency, proliferation, and epithelial–mesenchymal transition. Combinatorial therapy with EGF receptor and IGF-I receptor inhibitors prevents malignant progression. These results suggest that plasticity and recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for patients with TNBC. Significance: Currently, processes that mediate progression of otherwise indolent tumors are not well understood, making it difficult to accurately predict which patients with cancer are likely to relapse. Our findings reveal novel mechanisms of tumor phenotypic and gene expression plasticity that are mandated by microenvironmental factors, identifying novel therapeutic targets for patients with TNBC. Cancer Discov; 3(8); 922–35. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 826

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiological studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA. Here we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-κB). We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mammalian target of rapamycin complex 1 (mTORC1) inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, while combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports evaluation of aspirin and PI3K pathway inhibitors as combination therapy for targeting breast cancer.

Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer (LBC) cells and patient tumor specimens (LBC “instigators”) establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors (“responders”). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGF receptor 2+ tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically. Significance: Currently, processes that mediate progression of otherwise indolent tumors are not well understood, making it difficult to accurately predict which cancer patients are likely to relapse. Our findings highlight the macroenvironment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from adjuvant therapy. Cancer Discov; 2(12); 1150–65. ©2012 AACR. Read the Commentary on this article by DeFilippis and Tlsty, p. 1084 This article is highlighted in the In This Issue feature, p. 1065

Tumors have been increasingly recognized as organs with a complexity that approaches, and may even exceed, that of healthy tissues. When viewed from this perspective, the biology of a tumor can be understood only by studying tumor cell heterogeneity and the microenvironment that is constructed during the course of tumorigenesis and malignant progression. Recent work has revealed the existence of cancer stem cells, the "bugs", with the capacity for self-renewal and tumor propagation. In addition, it is now recognized that the tumor microenvironment, the "bed", plays a critical role in supporting cancer stem cells and also may promote neoplasia and malignant progression. The interdependence of the cell-intrinsic features of cancer, including the cancer stem cell "bugs" and the tumor microenvironment "bed", is only beginning to be understood. In this review, we highlight the rapidly evolving concepts about the interactions between tumor stem cells and their microenvironment, the insights gained from studying their normal tissue counterparts, and the questions and controversies surrounding this area of research, with an emphasis on breast and lung cancer. Finally, we address evidence supporting the notion that eliminating the bed as well as the bugs should lead to more effective and personalized cancer treatments that improve patient outcome.

Some breast cancer patients have no evidence of metastatic disease at the time of original diagnosis, yet ~30-40% of patients will experience recurrent breast cancer. Over 90% of breast cancer deaths occur due to distant spread of the disease, and bone is the most common site of breast cancer metastasis. Bisphosphonates, such as zoledronic acid (ZA), are used to treat patients with osteolytic disease, including metastatic breast cancer. ZA has been demonstrated to reduce disseminated tumor cells (DTCs) and breast cancer recurrence, but not all patients see this benefit and it is not yet clear what predicts benefit from adjuvant ZA treatment; the mechanism of action for this effect of ZA in reducing breast cancer remains undetermined. Here, we establish that ZA renders osteoclast progenitor cells (OCPs) tumor suppressive, primarily by directing their lineage potential. In instances where the OCP lineage potential was modulated via systemic or tumor-derived granulocyte-colony stimulating factor (G-CSF), OCPs did not differentiate into tumor-suppressive populations but instead into mature osteoclasts. High G-CSF was sufficient to abrogate the tumor-suppressive effect of ZA. Furthermore, we determined that in a subset of patient plasma samples from the AZURE clinical trial where stage II/III breast cancer patients were randomized to receive standard systemic treatment alone with adjuvant ZA treatment, patients who had baseline plasma G-CSF levels >23 pg/mL had reduced disease-free survival as assessed over a 10-year period post treatment initiation compared to patients with baseline plasma G-CSF levels Citation Format: Jessalyn M. Ubellacker, Ninib Baryawno, Nicolas Severe, Molly J. DeCristo, Jaclyn Sceneay, Marie-Therese Haider, Catherine S. Rhee, Yuanbo Qin, Ingunn Holen, Walter M. Gregory, Janet E. Brown, Robert E. Coleman, David T. Scadden, Sandra S. McAllister. Plasma G-CSF levels are predictive of lack of response to zoledronic acid treatment in reducing breast cancer recurrence [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B13.

Age is associated with increasing immune dysfunction that includes significant changes to both the innate and adaptive immune responses. These age-related changes could present limitations to the application of immunotherapy in breast cancer, as over 50% of patients are over 60 years old at diagnosis. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, but remains the only subtype that lacks any type of targeted therapy. Early results from clinical trials of anti-PD-1 therapy in metastatic TNBC patients have shown promise, with approximately 20% of patients demonstrating a partial or complete response. While TNBC incidence is similar in young and old patients, differences in disease progression and outcome with age suggest that treatment needs to be tailored to both age as well as breast cancer subtype. It is not known whether age impacts response to immunotherapy, particularly checkpoint blockade, in breast cancer. Using multiple preclinical models of age-dependent TNBC, we found that aged mice had significantly reduced ability to respond to either anti-PD-L1 or anti-CTLA4. Old mice showed decreased numbers of circulating CD4-positive and CD8-positive T cells, as well as NK cells. Furthermore, PD-1 expression was increased on these populations relative to young mice, suggesting increased immune cell exhaustion with age. The old mice also demonstrated increased central and effector memory T cells, but decreased naive T cells compared to young mice, consistent with reports from clinical studies of aged individuals. These findings suggest decreased ability to respond to neoantigens with age. Importantly, using high-content flow cytometric analyses and RNA-seq, we defined cellular and molecular mechanisms that correlate with response in young mice that are attenuated or lacking in old mice. Many of the differentially expressed genes that defined response to anti-CTLA4 in young mice and resistance to treatment in the old mice were also predictors of response to immunotherapy in cancer patients. Our data suggested novel combination therapies that, when combined with checkpoint blockade, improved responses and survival in old mice with TNBC. Our findings should suggest age-stratified treatments to ensure that TNBC patients see the most benefit possible and prevent overtreatment with futile and harsh chemotherapies. Citation Format: Sandra S. McAllister, Jaclyn Sceneay, Tyler Laszewski, Molly DeCristo, Jessalyn Ubellacker, Kristin Wilson, Yuanbo Qin, John Hutchinson. Efficacy of immunotherapy is attenuated with age in triple-negative breast cancer [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 1737.

Breast cancer mortality is principally due to tumor recurrence; however, the molecular mechanisms underlying this process are poorly understood. We now demonstrate that the suppressor of cytokine signaling protein SPSB1 is spontaneously upregulated during mammary tumor recurrence and is both necessary and sufficient to promote tumor recurrence in genetically engineered mouse models. The recurrence-promoting effects of SPSB1 result from its ability to protect cells from apoptosis induced by HER2/neu pathway inhibition or chemotherapy. This, in turn, is attributable to SPSB1 potentiation of c-MET signaling, such that preexisting SPSB1-overexpressing tumor cells are selected for following HER2/neu downregulation. Consistent with this, SPSB1 expression is positively correlated with c-MET activity in human breast cancers and with an increased risk of relapse in patients with breast cancer in a manner that is dependent upon c-MET activity. Our findings define a novel pathway that contributes to breast cancer recurrence and provide the first evidence implicating SPSB proteins in cancer.

Breast cancer is now the most frequently diagnosed cancer and leading cause of cancer death in women worldwide. Strategies targeting the primary tumour have markedly improved, but systemic treatments to prevent metastasis are less effective; metastatic disease remains the underlying cause of death in the majority of breast cancer patients who sucumb to their disease. The long latency period between initial treatment and eventual recurrence in some patients suggests that a tumour may both alter and respond to the host systemic environment in order to facilitate and sustain disease progression. Results from studies in animal models suggest that specific subtypes of breast cancer may direct metastasis through recruitment and activation of haematopoietic cells. In this review we focus on data implicating breast cancer as a systemic disease.

The causes for breast cancer recurrence in the form of metastatic disease and the reasons why less than 1% of disseminated tumor cells form metastases are unknown. A number of studies have demonstrated that the aggressive cancer cell population capable of driving metastasis feature properties of the epithelial-mesenchymal transition (EMT) and tumor initiation (TI). We previously reported novel mechanisms by which systemic and microenvironmental factors enrich tumors for EMT and TI genes and demonstrated that recurrence rates are not strictly due to tumor cell intrinsic properties (Castano et al., Cancer Discovery 2013). Using a preclinical model of breast cancer during the early phases of metastatic disease, when patients harbor disseminated tumor initiating cells (TICs) in the periphery at the time of their primary diagnosis (McAllister SS et al., Cell 2008), we recently made a surprising, and seemingly contrasting discovery. Specifically, we found that certain primary tumors can inhibit progression of disseminated TICs into overt tumors, maintaining them in the TI state and slowing their proliferation and differentiation to form tumor tissue mass. We established that the primary tumor induce mobilization of IL1beta-expressing monocytes that are recruited to sites where TICs reside. At those sites, signaling via the IL1 receptor maintains the EMT/TI state of the disseminated cells, thus promoting disease indolence. Importantly, surgical removal of the primary tumor enables TICs at the secondary sites to exit the EMT/TI state and to produce robustly growing tumors. Confirming our xenograft results, we established that stromal expression of IL1β in patient tumors is associated with poor outcome. Collectively, these data highlight the profound impact a primary tumor can exert on metastasizing cells - in this case, by altering the systemic environment to the detriment of secondary tumor growth. Moreover, our data highlight a central role for IL1β in modulating tumor cell plasticity and suggest it may provide a novel avenue for targeting recurrent disease. Citation Format: Zafira Castano, Christine L. Chaffer, Asaf Spiegel, Ayush Pant, Andrea L. Richardson, Ferenc Reinhardt, Timothy Marsh, Susanne Janssen, Ann M. Gifford, Robert A. Weinberg, Sandra S. McAllister. Understanding the systemic interactions between primary tumors and disseminated tumor initiating cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR13.

Aging is strongly associated with increased risk of developing breast cancer. Young age at diagnosis is typically associated with more aggressive disease, while breast cancer is generally more indolent in older women. Among the different breast cancer subtypes, triple negative breast cancer (TNBC) accounts for ∼15% of invasive breast cancer cases and TNBC patients have a poor prognosis due to lack of targeted therapies and limited sensitivity to chemotherapy. Emerging studies indicate that older women with TNBC have a better outcome than younger women for reasons that are unclear. Nevertheless, older patients tend to suffer co-morbidities associated with chemotherapy and often forego treatment, resulting in disease recurrence and a poor outcome for these patients. Treatment of TNBC therefore presents age-specific challenges. Here, we investigated how age affects the composition and function of pro-tumorigenic bone marrow cells using TNBC models that enable us to interrogate the impact of bone marrow derived cells on both tumor cells and the tumor microenvironment. Consistent with clinical observations, we found that disease progression was significantly more aggressive in young mice relative to aged mice. We identified age- and tumor-dependent molecular and functional changes to bone marrow derived hematopoietic cells that impact TNBC progression. A specific population of Sca1+/cKit- bone marrow cells that expresses CSF1R and secretes the growth factor granulin to support stromal activation and robust tumor growth in young mice, fails to promote tumor outgrowth in aged mice, resulting in maintenance of disease indolence. Importantly, bone marrow cells from young mice are sufficient to activate a tumor-supportive microenvironment and rescue tumor progression in aged mice. The results presented here indicate that hematopoietic age is an important determinant of TNBC aggressiveness and provide rationale for investigating age-stratified therapies designed to prevent activation of tumor-promoting bone marrow cells. Citation Format: Jaclyn Sceneay, Timothy Marsh, Irene Wong, Yuanbo Qin, Andreas Sjödin, Björn Nilsson, Sheila A. Stewart, Sandra S. McAllister. Hematopoietic age at onset of breast cancer dictates disease aggressiveness and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4400.

s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Cytotoxic chemotherapy is the only standard of care treatment currently available for patients with triple negative breast cancer (TNBC) because this breast cancer subtype lacks expression of therapeutic targets, namely, estrogen receptor, progesterone receptor, and amplification of Her2. Despite the efficacy of neoadjuvant chemotherapy for some TNBC patients, approximately 60-80% of TNBC patients will still present with local and distant disease after therapy, and these patients suffer poor prognosis. In addition to direct effects on tumor cells, systemic chemotherapy also has the potential to impact patient physiology. However, the implications of tumor-cell independent effects of chemotherapy for metastatic progression are not well understood. We are using mouse models of TNBC to study the effects of chemotherapy on host physiology and how these effects impact metastasis. Treatment of TNBC tumor-bearing mice with doxorubicin (A), cyclophosphamide (C), and paclitaxel (T) decreased overall bone marrow cell counts; although, the degree of decrease and subsequent recovery of individual bone marrow and circulating cell populations varied among populations. Notably, parallel experiments in tumor-free mice demonstrated that chemotherapy-dependent effects on the bone marrow and circulating cell populations dominate tumor-dependent effects. These findings highlight the potential implications of the physiological effects of chemotherapy on metastasis and response to therapy. Citation Format: Molly J. DeCristo, Sandra S. McAllister. Systemic effects of AC-T chemotherapy in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A66.

Recurrent disease affects approximately 30% of breast cancer patients but, unfortunately, by the time overt metastases are detected, it is no longer possible to treat breast cancer patients with curative intent. Clinical evidence indicates that in women who are diagnosed with metastatic breast cancer, tumor cells disseminate from the primary tumor prior to surgical resection but can remain indolent for varying periods of time before the patient becomes symptomatic. The reasons why certain disseminated cells remain indolent while others erupt into malignant tumors remain unknown; it is therefore currently difficult to predict with any degree of accuracy which patients will experience disease relapse. Our lab uses various mouse and xenograft models of breast cancer progression to study the early phases of metastatic disease when patients harbor indolent disseminated tumor cells in the periphery at the time of their primary diagnosis. Using triple-negative models, we previously showed that certain tumor systemically support the outgrowth of distant, otherwise indolent disseminated tumors, doing so by secreting cytokines into the host circulation and activating bone marrow hematopoietic cells to become pro-tumorigenic (McAllister, et al., Cell, 2008; Elkabets, et al., JCI, 2011; Castano, et al, Ca Disc, 2013). Using models of luminal breast cancer, we discovered that certain tumors promote the outgrowth of distant tumors by supporting angiogenesis (Kuznetsov, et al., Ca Disc, 2012). More recently, we have begun to identify the properties of disseminated tumor cells that are poised to respond to systemic factors and importantly, the phenotype of those that do not respond to systemic regulation. We have learned that certain systemic factors (cytokines and hematopoietic cells) elicited by primary breast tumors can sustain tumor-initiating properties of disseminated cells. Our findings highlight the systemic environment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from neoadjuvant or adjuvant therapy. Moreover, we hope that this work will pave the way to designing new therapies that can be delivered at a time when what has previously been an inexorable progression to terminal metastatic disease may be prevented. Citation Format: Sandra S. McAllister. Systemic regulation of breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA07.

s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Bisphosphonates, such as zoledronic acid (ZOL), are bone microenvironment modulators that work primarily by inhibiting the activity of bone resorbing osteoclasts and have demonstrated ability to reduce breast cancer risk and recurrence in the bone in clinical trials. Previous work in our laboratory has demonstrated the ability of tumor-derived factors (osteopontin) to activate pro-tumorigenic bone marrow hematopoietic cells, which subsequently promote tumor outgrowth. The aim of this current study is to determine whether ZOL inhibits the impact of such tumor-derived paracrine factors on the bone microenvironment, thus reducing metastatic outgrowth in the bone. A human bone-tropic breast cancer cell line (MDA-MB-231 BO2) was introduced into NU/J mice via intracardiac (IC) and intratibial (IT) injections 72 hours after treating the mice with ZOL. Cell seeding was assessed at 24 hours by CM-DiL staining, and subsequent tumor outgrowth was monitored weekly by bioluminescence. One ZOL pre-dose was sufficient to decrease both tumor cell seeding (via IC) and colonization (via IT) relative to the robust tumor outgrowth observed in mice treated with vehicle control. Additionally, subcutaneous ad-mixtures of MDA-MB-231 BO2 cells with bone marrow cells from ZOL-treated mice demonstrated a decreased incidence of tumor formation relative to control bone marrow cells, indicating that the tumor inhibitory effect of ZOL occurs via modulations to the bone marrow hematopoietic microenvironment. A second MDA-MB-231 bone-tropic cell line similar to the BO2 line, but with higher expression levels of CXCL1, G-CSF, GM-CSF and IL8, was not inhibited by ZOL in the IC and IT bone metastasis models. These results implicate the role of certain tumor-derived paracrine factors in mediating resistance to ZOL treatment. These findings are important for elucidating the therapeutic potential of bisphosphonates for treating at-risk patients in the adjuvant setting before they develop incurable metastatic disease. Citation Format: Jessalyn M. Ubellacker, Sandra S. McAllister. Elucidating the impact of zoledronic acid on the bone microenvironment: Implications for breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A67.

Little is known about the mechanisms that cause indolent tumors – such as micrometastases, occult primary tumors, or minimal residual disease – to erupt into overt, malignant cancers. As a result, predicting which patients are likely to experience disease relapse and treating patients with metastatic disease has been frustratingly limited. We developed an in vivo xenograft model system that provided us with fundamental insights into processes that govern indolent tumor growth and represented a new paradigm for translational cancer research. We learned that systemic endocrine factors and circulating bone marrow-derived cells support the acquisition of malignant traits by otherwise indolent tumors. As a result, we now think of cancer as a systemic disease by which tumors actively perturb as well as respond to the host systemic environment. First, we found that certain human breast carcinoma cell lines (we term “instigators”) facilitate the growth of otherwise-indolent tumor cells (we term “responders”) located at distant anatomical sites within host mice – a process we term “systemic instigation”. Second, systemic instigation is accompanied by incorporation of bone marrow-derived cells into the stroma of the distant once-indolent tumors. Importantly, bone marrow cells (BMCs) of hosts bearing instigating tumors are functionally activated in the marrow prior to their mobilization into the circulation. Third, instigating tumor-derived osteopontin (OPN), a cytokine that is elevated in the plasma of patients with metastatic cancers and is predicitive of poor outcome, is necessary but not sufficient for systemic instigation. Although there may be alternative explanations, this systemic communication between tumors might explain why patients diagnosed with one malignant neoplasm are at an increased risk of presenting with multiple, independent primary cancers or why patients with recurrent disease often present with multiple metastases that appear to arise suddenly and synchronously. In this review, I address the methods by which the systemic instigation model was established, what we’ve learned by using this model, the implications of our studies, and some of the questions that have yet to be answered.

The majority of breast neoplasms are diagnosed in patients who are between the ages of 65 and 80. Although tumor growth is generally less aggressive and metastasis is less prevalent in older patients than in younger patients, elderly patients suffer the highest mortality and morbidity associated with breast cancer and breast cancer treatment. The reasons for age-dependent differences in incidence and aggressiveness of breast cancer are not understood. Treatment of breast cancer patients therefore presents age-specific challenges. We recently established mouse and human xenograft breast tumor models that mimic the age dependent disease progression that is observed in breast cancer patients, whereby tumor cells implanted into aged mice had lower incidence and slower growth kinetics than when implanted into young mice. Moreover, alpha-smooth muscle actin-positive myofibroblasts within the tumor microenvironment were significantly less prevalent in the tumors from aged mice than in size-matched tumors from young mice. We discovered that bone marrow derived cells from aged mice were significantly less effective in supporting breast cancer progression than the counterpart cells from young mice. Strikingly, bone marrow derived cells from young mice rescued tumor growth and stromal desmoplasia in aged mice. We are elucidating some of the molecular and phenotypic differences between young and aged tumor-promoting bone marrow cells. For example, we found that bone marrow cells from aged mice express lower levels of the cytokine, granulin, which is known to activate normal mammary fibroblasts in young mice. Our findings indicate that the age of the host hematopoietic system is a powerful determinant of breast tumor progression. Defining functional and molecular differences between tumor supportive hematopoietic cells in young and aged hosts should lay a foundation for further work in this relatively uncharted area of cancer research. Understanding tumor support mechanisms that exist in young and aged hosts should suggest new ideas for preclinical development of age-stratified breast cancer therapies. Citation Format: Timothy Marsh, Irene Wong, Amey Barakat, Yuanbo Qin, Sandra S. McAllister. Hematopoietic system aging has profound effects on the breast tumor microenvironment and cancer progression. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR11. doi:10.1158/1538-7445.CHTME14-PR11

By the time metastatic breast cancer is detected, patients are no longer treated with curative intent. Women with triple-negative breast cancer (TNBC) are particularly at risk for early recurrence. Clinical evidence indicates that in women with metastatic TNBC, the primary tumor and disseminated cells co-existed for an indefinite period of time. We demonstrate that outgrowth of distant tumors that would otherwise remain indolent in cancer-free mice is accelerated in mice with TNBC. Systemic signals provided by TNBCs cause formation of a tumor-supportive microenvironment at distant tumor sites. This microenvironment is enriched for EGF and IGF-1, which convert indolent tumors to a malignant state, defined by expression of factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Inhibition of both EGFR and IGF1R maintains these tumors in the indolent state. These results suggest that recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for TNBC patients. Citation Format: Zafira Castano, Tim Marsh, Ramya Tadipatri, Hanna S. Kuznetsov, Fatima Al-Shahrour, Mahanaz Paktinat, April Greene-Colozzi, Bjorn Nilsson, Andrea L. Richardson, Sandra S. McAllister. Systemic and stromal support of triple-negative breast cancer progression is prevented by EGF and IGF-1 inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C94.

Breast cancer recurrence rates are variable, suggesting that tumor cells disseminate from primary sites at an early stage but remain indolent for extended periods of time before progressing to symptomatic disease. Little is known about mechanisms that cause otherwise dormant tumors to become overt cancers, making it difficult to predict which breast cancer patients are likely to relapse and therefore likely to benefit from preemptive therapy. To address these challenges, we use a human breast tumor xenograft model in which aggressively growing breast cancers (“instigators”) promote the growth of otherwise indolent breast cancers (“responders”) that have disseminated to distant sites. These pro-tumorigenic systemic effects are established by instigating tumors, in large part, through their mobilization of bone marrow cells (BMCs) that are subsequently recruited to the responding breast tumors, where they create a tumor-supportive microenvironment. We report here that the mechanisms of establishing tumor-supportive systemic environments depend upon the molecular-histopathological subtype of the instigating breast cancer. Instigating cell lines and primary tumor surgical specimens, derived from patients with either triple-negative breast cancer (TNBC) or luminal breast cancer (LBC), established different subtype-specific systemic environments that impinged in different ways upon the malignant features of responding tumor cells and their microenvironment. In particular, the TNBC-induced responding tumor microenvironment was characterized by elevated levels of epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF1). EGF and IGF acted on responding tumor cells in a paracrine manner to induce expression of factors that play a role in tumor-initiation, embryonic stem cell maintenance, and epithelial-mesenchymal transition. Pharmacological administration of EGFR and IGF1R inhibitors prevented responding tumors from acquiring the TNBC-induced malignant features, thus maintaining them in an indolent state. Our results demonstrate that microenvironmental composition, as well as tumor cell phenotypic plasticity and molecular properties, are strongly influenced by the host systemic macroenvironment. Our work suggests that the crosstalk between disseminated tumor cells and their microenvironment can be exploited therapeutically to improve outcomes for patients with TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4861. doi:1538-7445.AM2012-4861