The Innate Immune System and TRAIL-BCL-XL Axis Mediate a Sex Bias in Lung Cancer and Confer a Therapeutic Vulnerability in Females.

There is a significant sex-bias in lung cancer with males showing increased mortality compared to females. A better mechanistic understanding of these differences could help identify therapeutic targets to personalize cancer therapies to each sex. After observing a clear sex-bias in humanized mice, with male patient-derived xenograft (PDX) lung tumors being more progressive and deadlier than female PDX lung tumors, we identified mouse tumor models of lung cancer with the same sex-bias. This sex-bias was not observed in models of breast, colon, melanoma, and renal cancers. In vivo, the sex-bias in growth and lethality required intact ovaries, functional innate natural killer (NK) cells and monocytes/macrophages, and the activating receptor NKG2D. Ex vivo cell culture models were sensitized to the anti-cancer effects of NKG2D-mediated NK cell and macrophage killing through the TRAIL-BCL-XL axis when cultured with serum from female mice with intact ovaries. In both flank and orthotopic models, the BCL-XL inhibitor navitoclax (ABT-263) improved tumor growth control in female mice and required NK cells, macrophages, and the TRAIL signaling pathway. This research suggests that navitoclax and TRAIL pathway agonists could be used as a personalized therapy to improve outcomes in women with lung cancer.

Proteomic analysis of dorsal root ganglia in a mouse model of paclitaxel-induced neuropathic pain.

Paclitaxel is a chemotherapy drug widely used for the treatment of various cancers based on its ability to potently stabilize cellular microtubules and block division in cancer cells. Paclitaxel-based treatment, however, accumulates in peripheral system sensory neurons and leads to a high incidence rate (over 50%) of chemotherapy induced peripheral neuropathy in patients. Using an established preclinical model of paclitaxel-induced peripheral neuropathy (PIPN), we examined proteomic changes in dorsal root ganglia (DRG) of adult male mice that were treated with paclitaxel (8 mg/kg, at 4 injections every other day) relative to vehicle-treated mice. High throughput proteomics based on liquid chromatography electrospray ionization mass spectrometry identified 165 significantly altered proteins in lumbar DRG. Gene ontology enrichment and bioinformatic analysis revealed an effect of paclitaxel on pathways for mitochondrial regulation, axonal function, and inflammatory purinergic signaling as well as microtubule activity. These findings provide insight into molecular mechanisms that can contribute to PIPN in patients.

Lymph node-targeting adjuvant/neoantigen-codelivering vaccines for combination glioblastoma radioimmunotherapy.

Glioblastoma multiforme (GBM) is the most common and lethal type of adult brain cancer. Current GBM standard of care, including radiotherapy, often ends up with cancer recurrence, resulting in limited long-term survival benefits for GBM patients. Immunotherapy, such as immune checkpoint blockade (ICB), has thus far shown limited clinical benefit for GBM patients. Therapeutic vaccines hold great potential to elicit anti-cancer adaptive immunity, which can be synergistically combined with ICB and radiotherapy. Peptide vaccines are attractive for their ease of manufacturing and stability, but their therapeutic efficacy has been limited due to poor vaccine co-delivery and the limited ability of monovalent antigen vaccines to prevent tumor immune evasion. To address these challenges, here, we report GBM radioimmunotherapy that combines radiotherapy, ICB, and multivalent lymph-node-targeting adjuvant/antigen-codelivering albumin-binding vaccines (AAco-AlbiVax). Specifically, to codeliver peptide neoantigens and adjuvant CpG to lymph nodes (LNs), we developed AAco-AlbiVax based on a Y-shaped DNA scaffold that was site-specifically conjugated with CpG, peptide neoantigens, and albumin-binding maleimide-modified Evans blue derivative (MEB). As a result, these vaccines elicited antitumor immunity including neoantigen-specific CD8+ T cell responses in mice. In orthotopic GBM mice, the combination of AAco-AlbiVax, ICB, and fractionated radiation enhanced GBM therapeutic efficacy. However, radioimmunotherapy only trended more efficacious over radiotherapy alone. Taken together, these studies underscore the great potential of radioimmunotherapy for GBM, and future optimization of treatment dosing and scheduling would improve the therapeutic efficacy.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

Autophagy-Dependent Sensitization of Triple-Negative Breast Cancer Models to Topoisomerase II Poisons by Inhibition of the Nucleosome Remodeling Factor.

Epigenetic regulators can modulate the effects of cancer therapeutics. To further these observations, we discovered that the bromodomain PHD finger transcription factor subunit (BPTF) of the nucleosome remodeling factor (NURF) promotes resistance to doxorubicin, etoposide, and paclitaxel in the 4T1 breast tumor cell line. BPTF functions in promoting resistance to doxorubicin and etoposide, but not paclitaxel, and may be selective to cancer cells, as a similar effect was not observed in embryonic stem cells. Sensitization to doxorubicin and etoposide with BPTF knockdown (KD) was associated with increased DNA damage, topoisomerase II (TOP2) crosslinking and autophagy; however, there was only a modest increase in apoptosis and no increase in senescence. Sensitization to doxorubicin was confirmed in vivo with the syngeneic 4T1 breast tumor model using both genetic and pharmacologic inhibition of BPTF. The effects of BPTF inhibition in vivo are autophagy dependent, based on genetic autophagy inhibition. Finally, treatment of 4T1, 66cl4, 4T07, MDA-MB-231, but not ER-positive 67NR and MCF7 breast cancer cells with the selective BPTF bromodomain inhibitor, AU1, recapitulates genetic BPTF inhibition, including in vitro sensitization to doxorubicin, increased TOP2-DNA crosslinks and DNA damage. Taken together, these studies demonstrate that BPTF provides resistance to the antitumor activity of TOP2 poisons, preventing the resolution of TOP2 crosslinking and associated autophagy. These studies suggest that BPTF can be targeted with small-molecule inhibitors to enhance the effectiveness of TOP2-targeted cancer chemotherapeutic drugs. IMPLICATIONS: These studies suggest NURF can be inhibited pharmacologically as a viable strategy to improve chemotherapy effectiveness.

Unexpected PD-L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies.

Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor-enriched death receptor-5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase-II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD-L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase-8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD-L1 stability on tumor cell surface. Targeting DR5-ROCK1-PD-L1 axis markedly increases immune effector T-cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism.

Regulatory T Cells Support Breast Cancer Progression by Opposing IFN-γ-Dependent Functional Reprogramming of Myeloid Cells.

Regulatory T (Treg) cell infiltration of solid tumors often correlates with poor prognosis, but their tumor-suppressive function lacks mechanistic understanding. Through a combination of transgenic mice, cell fate mapping, adoptive transfer, and co-injection strategies, we demonstrate that Treg cell ablation-dependent anti-tumor effects in murine breast cancer require intratumoral recruitment of CCR2+ inflammatory monocytes, which primarily differentiate into tumor-associated macrophages (TAMs), and lead to reprogramming of their function in an IFN-γ-dependent manner. Furthermore, transcriptomic signatures from murine TAMs in Treg cell-ablated conditions correlate with increased overall survival in human breast cancer. Our studies highlight the strong myeloid dependency of breast cancer and provide the basis for the development of therapeutic strategies based on manipulation of the IFN-γ signaling pathway in monocytes.

Local Targeting of Lung-Tumor-Associated Macrophages with Pulmonary Delivery of a CSF-1R Inhibitor for the Treatment of Breast Cancer Lung Metastases.

The lungs are major sites of metastases for several cancer types, including breast cancer (BC). Prognosis and quality of life of BC patients that develop pulmonary metastases are negatively impacted. The development of strategies to slow the growth and relieve the symptoms of BC lung metastases (BCLM) is thus an important goal in the management of BC. However, systemically administered first line small molecule chemotherapeutics have poor pharmacokinetic profiles and biodistribution to the lungs and significant off-target toxicity, severely compromising their effectiveness. In this work, we propose the local delivery of add-on immunotherapy to the lungs to support first line chemotherapy treatment of advanced BC. In a syngeneic murine model of BCLM, we show that local pulmonary administration (p.a.) of PLX-3397 (PLX), a colony-stimulating factor 1 receptor inhibitor (CSF-1Ri), is capable of overcoming physiological barriers of the lung epithelium, penetrating the tumor microenvironment (TME), and decreasing phosphorylation of CSF-1 receptors, as shown by the Western blot of lung tumor nodules. That inhibition is accompanied by an overall decrease in the abundance of protumorigenic (M2-like) macrophages in the TME, with a concomitant increase in the amount of antitumor (M1-like) macrophages when compared to the vehicle-treated control. These effects with PLX (p.a.) were achieved using a much smaller dose (1 mg/kg, every other day) compared to the systemic doses typically used in preclinical studies (40-800 mg/kg/day). As an additive in combination with intravenous (i.v.) administration of paclitaxel (PTX), PLX (p.a.) leads to a decrease in tumor burden without additional toxicity. These results suggested that the proposed immunochemotherapy, with regional pulmonary delivery of PLX along with the i.v. standard of care chemotherapy, may lead to new opportunities to improve treatment, quality of life, and survival of patients with BCLM.

Brain Metastasis Cell Lines Panel: A Public Resource of Organotropic Cell Lines.

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood-brain barrier, blood-tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

Tracing bone marrow-derived microglia in brain metastatic tumors.

Microglia are the resident macrophages in the central nervous system (CNS), and they constitute 15-20% of the total glial populations. They have wide developmental and protective functions during brain injury, infection and tumorigenesis. Originally thought to derive from postnatal hematopoietic progenitors, it has recently been demonstrated that microglia originate from primitive myeloid progenitor cells that arise during early development from the embryonic yolk sac. Circulating monocytes infiltrate the CNS upon inflammatory conditions, such as cancer, primarily differentiating into macrophages and dendritic cells. Both resident and recruited microglia respond to environmental cues and actively participate in pathogenic processes, albeit their transcriptomic profiles contain significant differences suggesting distinctive roles. Metastatic brain tumors are the most common intracranial neoplasm in adults, with an estimate incidence 10 times higher than all primary brain neoplasms combined, and with dismal prognosis. Microglia is a major immune population associated with brain metastatic tumors in patients. They are proposed to play multiple, and sometimes opposing roles, in tumor progression. However, our ability to evaluate individual contribution of resident and recruited populations is hindered by the fact that they express overlapping sets of surface markers. Tracking and interrogating tissue-resident vs recruited microglia in the brain tumor microenvironment becomes critical to dissect their respective roles and gain a better understanding of the mechanism governing their interaction. In this chapter, we describe the utilization of genetic reporter mice to identify recruited brain microglia, offer a comparison between the genetic method and the most widely used flow cytometric approach, and discuss potential downstream applications to interrogate BMDM function in brain metastatic disease.

Regulatory T Cells Control the Switch From in situ to Invasive Breast Cancer.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of breast cancer, and it only progresses to invasive breast cancer in around 40% of patients. While immune infiltrates have been observed in these early cancer lesions, their potential prognostic value is still unclear. Regulatory T (Treg) cells accumulate in advanced breast cancers, and predict poor outcome. We have shown before that ablation of Treg cells in established tumors leads to significant decrease in primary and metastatic tumor burden. In this work, we sought to investigate Treg cell function in the progression from non-invasive to invasive breast cancer lesions. To this end, we used the murine mammary tumor virus polyoma middle T (MMTV-PyMT) murine model of spontaneous, stage-wise breast carcinogenesis crossed to Foxp3 DTR knock in mice, allowing Treg cell ablation by administration of diphtheria toxin. Transient targeting of Treg cells at the in situ carcinoma stage resulted in a significant increase in the number of tumor-bearing mammary glands and size of growing tumors compared with control mice. Whole mammary gland mounts and histological examination confirmed larger emergent tumor area in Treg cell-ablated mice, and revealed that these tumors were characterized by a more advanced tumor staging, with presence of early invasion, increased desmoplasia and collagen deposition. Furthermore, Treg cell ablation increased the percentage of cancer stem/progenitor cells in the mammary compartment. Interestingly, Treg cell ablation resulted in increased inflammatory cytokines IL-4 and IL-5 with a concomitant reduction in classically activated tumor associated macrophages. This TH2-biased immune regulatory mammary inflammation was consistent with the enhancement in tumor promotion that we observed. Overall, our study demonstrates that Treg cells oppose breast cancer progression at early stages, raising a cautionary note regarding the consideration of immune intervention targeted at boosting immune responses for DCIS.

Preexisting Commensal Dysbiosis Is a Host-Intrinsic Regulator of Tissue Inflammation and Tumor Cell Dissemination in Hormone Receptor-Positive Breast Cancer.

It is unknown why some patients with hormone receptor-positive (HR+) breast cancer present with more aggressive and invasive disease. Metastatic dissemination occurs early in disease and is facilitated by cross-talk between the tumor and tissue environment, suggesting that undefined host-intrinsic factors enhance early dissemination and the probability of developing metastatic disease. Here, we have identified commensal dysbiosis as a host-intrinsic factor associated with metastatic dissemination. Using a mouse model of HR+ mammary cancer, we demonstrate that a preestablished disruption of commensal homeostasis results in enhanced circulating tumor cells and subsequent dissemination to the tumor-draining lymph nodes and lungs. Commensal dysbiosis promoted early inflammation within the mammary gland that was sustained during HR+ mammary tumor progression. Furthermore, dysbiosis enhanced fibrosis and collagen deposition both systemically and locally within the tumor microenvironment and induced significant myeloid infiltration into the mammary gland and breast tumor. These effects were recapitulated both by directly targeting gut microbes using nonabsorbable antibiotics and by fecal microbiota transplantation of dysbiotic cecal contents, demonstrating the direct impact of gut dysbiosis on mammary tumor dissemination. This study identifies dysbiosis as a preexisting, host-intrinsic regulator of tissue inflammation, myeloid recruitment, fibrosis, and dissemination of tumor cells in HR+ breast cancer. SIGNIFICANCE: Identification of commensal dysbiosis as a host-intrinsic factor mediating evolution of metastatic breast cancer allows for development of interventions or diagnostic tools for patients at highest risk for developing metastatic disease.See related commentary by Ingman, p. 3539.

Tumor-Associated Macrophage Isolation and In Vivo Analysis of Their Tumor-Promoting Activity.

Characterization of individual cell populations from the tumor microenvironment is critical to understand their functional contribution to tumor progression. Magnetic bead enrichment and fluorescence-activated cell sorting (FACS) allow for the isolation of specific cell types that can be used in downstream applications, including in vitro and in vivo functional studies and molecular profiling. In this chapter, we describe the process of isolation of tumor-associated macrophages (TAMs) from primary murine breast tumors subsequent to therapeutic or experimental intervention. Additionally, we further detail how to analyze their ability to support tumor cell growth by co-injecting isolated TAMs with tumor cells orthotopically into the mammary gland of immune-deficient hosts, and monitoring tumor progression by live imaging and caliper measurement.

Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer.

Regulatory T (Treg) cells reside in lymphoid organs and barrier tissues where they control different types of inflammatory responses. Treg cells are also found in human cancers, and studies in animal models suggest that they contribute to cancer progression. However, properties of human intratumoral Treg cells and those present in corresponding normal tissue remain largely unknown. Here, we analyzed features of Treg cells in untreated human breast carcinomas, normal mammary gland, and peripheral blood. Tumor-resident Treg cells were potently suppressive and their gene-expression pattern resembled that of normal breast tissue, but not of activated peripheral blood Treg cells. Nevertheless, a number of cytokine and chemokine receptor genes, most notably CCR8, were upregulated in tumor-resident Treg cells in comparison to normal tissue-resident ones. Our studies suggest that targeting CCR8 for the depletion of tumor-resident Treg cells might represent a promising immunotherapeutic approach for the treatment of breast cancer.

TREG Cells in Cancer: Beyond Classical Immunological Control.

A prerequisite for tumor evolution toward a malignant state is the establishment of cell intrinsic and extrinsic mechanisms of immune suppression (Hanahan and Weinberg, 2000, 2011; Schreiber, Old, and Smyth, 2011). Widespread recruitment of Foxp3+ regulatory T cells (TREG) is a prevailing means to dampen antitumor immunity. Advances in the characterization of TREG cell heterogeneity and physiological function of tissue resident TREG cells unfold new possibilities for nontraditional tumor-promoting functions of intratumoral TREG cells. This review will focus on the nonclassical function of TREG cells and their implicancies for cancer biology and treatment.

A Single miRNA-mRNA Interaction Affects the Immune Response in a Context- and Cell-Type-Specific Manner.

MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotypes and controls responses to extracellular stimuli. Although a single miRNA can regulate expression of hundreds of target genes, it is unclear whether any of its distinct biological functions can be due to the regulation of a single target. To explore in vivo the function of a single miRNA-mRNA interaction, we mutated the 3' UTR of a major miR-155 target (SOCS1) to specifically disrupt its regulation by miR-155. We found that under physiologic conditions and during autoimmune inflammation or viral infection, some immunological functions of miR-155 were fully or largely attributable to the regulation of SOCS1, whereas others could be accounted only partially or not at all by this interaction. Our data suggest that the role of a single miRNA-mRNA interaction is dependent on cell type and biological context.

Transient regulatory T cell ablation deters oncogene-driven breast cancer and enhances radiotherapy.

Rational combinatorial therapeutic strategies have proven beneficial for the management of cancer. Recent success of checkpoint blockade in highly immunogenic tumors has renewed interest in immunotherapy. Regulatory T (T reg) cells densely populate solid tumors, which may promote progression through suppressing anti-tumor immune responses. We investigated the role of T reg cells in murine mammary carcinogenesis using an orthotopic, polyoma middle-T antigen-driven model in Foxp3(DTR) knockin mice. T reg cell ablation resulted in significant determent of primary and metastatic tumor progression. Importantly, short-term ablation of T reg cells in advanced spontaneous tumors led to extensive apoptotic tumor cell death. This anti-tumor activity was dependent on IFN-γ and CD4(+) T cells but not on NK or CD8(+) T cells. Combination of T reg cell ablation with CTLA-4 or PD-1/PD-L1 blockade did not affect tumor growth or improve the therapeutic effect attained by T reg cell ablation alone. However, T reg cell targeting jointly with tumor irradiation significantly reduced tumor burden and improved overall survival. Together, our results demonstrate a major tumor-promoting role of T reg cells in an autochthonous model of tumorigenesis, and they reveal the potential therapeutic value of combining transient T reg cell ablation with radiotherapy for the management of poorly immunogenic, aggressive malignancies.

IL-2-dependent adaptive control of NK cell homeostasis.

Activation and expansion of T and B lymphocytes and myeloid cells are controlled by Foxp3(+) regulatory T cells (T reg cells), and their deficiency results in a fatal lympho- and myeloproliferative syndrome. A role for T reg cells in the homeostasis of innate lymphocyte lineages remained unknown. Here, we report that T reg cells restrained the expansion of immature CD127(+) NK cells, which had the unique ability to up-regulate the IL2Rα (CD25) in response to the proinflammatory cytokine IL-12. In addition, we observed the preferential accumulation of CD127(+) NK cells in mice bearing progressing tumors or suffering from chronic viral infection. CD127(+) NK cells expanded in an IL-2-dependent manner upon T reg cell depletion and were able to give rise to mature NK cells, indicating that the latter can develop through a CD25(+) intermediate stage. Thus, T reg cells restrain the IL-2-dependent CD4(+) T cell help for CD127(+) immature NK cells. These findings highlight the adaptive control of innate lymphocyte homeostasis.

Treg cells in cancer: a case of multiple personality disorder.

Foxp3(+) RORγt-expressing T cells expand in colorectal cancer and contribute to pathogenesis in a mouse model of polyposis.