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3D scaffolds mimicking the environment in the primary tumor or metastatic organs can deconstruct complex niche signals and facilitate the study of cancer progression and metastasis. Here, we reported that a subcutaneous 3D scaffold implant acted as a lung-mimicking dormant metastatic niche in mouse models of metastatic breast cancer, recruiting lung-tropic circulating tumor cells yet suppressing their growth through potent in situ antitumor immunity. We compared it with the immunosuppressive lungs developing lethal metastases and the dormant lungs suppressing tumor growth derived from breast cancer models with varying tumor aggressiveness and host immunity. Our data suggested that breast cancer-induced Gr1+CD11b+Ly6G+ granulocytic myeloid cells (neutrophils) infiltrated the scaffold implants and lungs, secreting the same signal to facilitate the metastatic seeding of lung-tropic cancer cells in these two types of niches. However, circulating neutrophils with opposing phenotypes and functions (N1 and N2) were selectively recruited and enriched in the dormant scaffolds/lungs and immunosuppressive lungs, respectively, responding to two distinct groups of chemoattractants. N1 or N2 neutrophils established activated or suppressive immune environments in the metastatic niches, directing different fates of cancer cells. The clinical relevance of these scientific findings was validated by the strong positive correlation of a high N1-to-N2 neutrophil chemoattractant ratio with a low-grade primary tumor, a low metastases incidence, and a better prognosis in breast cancer patients. Overall, our study revealed the multifaceted roles of neutrophils in regulating lung metastasis and underscored the importance of N1 neutrophils in driving breast cancer metastatic dormancy in the lungs, inspiring next-generation immunotherapy. Citation Format: Jing Wang, Ramon Ocadiz-Ruiz, Matthew Hall, Grace Bushnell, Sophia Orbach, Joseph Decker, Ravi Raghani, Yining Zhang, Aaron Morris, Jacqueline Jeruss, Lonnie Shea. A lung-mimicking synthetic metastatic niche reveals N1 neutrophils drive breast cancer metastatic dormancy in the lungs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB347.

e14552 Background: Recent advances in the field of cancer immunology have highlighted the importance of the immune system for eliminating tumors. Numerous studies have shown that tumor-infiltrating immune cells such as antigen-presenting cells (APCs), T cells, and natural killer (NK) cells play critical roles in tumor control. However, the inflammatory anti-tumor immune response is counteracted by the induction of immune regulatory mechanisms within the tumor microenvironment (TME). These findings have led to the development of immune-targeted therapies, which are aimed at activating anti-tumor immune signaling pathways and enhancing anti-tumor immune function. While immunotherapies, have revolutionized the treatment of several solid tumors and leukemias, at best response rates remain low at 25%-30%, and a portion of patients eventually develop resistance to therapy leading to disease progression and mortality. Methods: We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic- co-glycolic acid) (PLGA) nanoparticles, ONP-302, free from drugs or other bioactive agents, for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302 nanoparticles, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. The efficacy of ONP-302 nanoparticles at inducing an anti-tumor immune response was evaluated using syngeneic mouse tumor models. Results: ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. Conclusions: These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

e14555 Background: Metastasis is responsible for most cancer-related deaths and depends on the formation of a metastatic niche at distal sites. The metastatic niche consists primarily of immunosuppressive myeloid cells, such as neutrophils and monocytes. These cells are recruited to distal sites in response to cancer-induced immune dysregulation and promote the recruitment and survival of disseminated tumor cells. We have employed drug-free poly(lactide-co-glycolide) (PLGA) nanoparticles, ONP-302, to modulate myeloid cell phenotypes and trafficking to disrupt the formation of the metastatic niche. The anti-metastatic efficacy of ONP-302 nanoparticles was evaluated in a translationally relevant mouse model of orthotopic triple-negative breast cancer. Methods: ONP-302 nanoparticles were evaluated in the syngeneic 4T1 model of triple-negative breast cancer in female BALB/c or RAG1KO BALB/c mice. ONP-302 nanoparticles intravenously injected into tumor-bearing mice every 3 days starting 1 day after tumor inoculation. Lungs were extracted 14 and 21 days after inoculation for analysis by single cell RNA sequencing and quantification of metastases by luminescent imaging, respectively. In resection studies, primary tumors were resected 11 days after inoculation and tissues were extracted 42 days after resection for quantification of metastases. Results: ONP-302 nanoparticles significantly reduced primary tumor growth and completely inhibited the formation of lung metastases. ONP-302 was also effective at suppressing metastasis when applied following surgical resection. In mice which received ONP-302 after surgical resection, lung metastases were undetectable up to 42 days after resection, while 44% of control mice developed metastases. These findings suggest that ONP-302 targets both primary tumor growth and metastatic colonization. Single cell RNA sequencing was employed to analyze ONP-302 modulation of the lung metastatic niche. The lungs of ONP-302 treated mice had lower neutrophil infiltration and a higher proportion of monocytes and dendritic cells compared to control mice. Gene expression pathways associated with inflammatory interferon and tumor necrosis factor responses were upregulated in neutrophils, monocytes, and dendritic cells from ONP-302 treated mice. The upregulation of these pathways suggests that ONP-302 treatment prevents the formation of an immunosuppressive niche and promotes anti-tumor cytotoxicity. Adaptive immunity was essential for ONP-302 efficacy, as T and B cell deficient RAG1KO mice demonstrated complete loss of ONP-302 efficacy both in slowing primary tumor growth and preventing metastasis. Conclusions: Our findings demonstrate that our drug-free PLG nanoparticles, ONP-302, impede cancer metastasis as a monotherapy by inhibiting the accumulation of immunosuppressive myeloid cells.

SummaryData on human skin fibroblast transcriptional responses to external cues were used to reconstruct dynamic gene regulatory networks. The goal of the reconstruction was to determine dynamic network interactions (quantitative predictive relationships of mutual regulatory influences of and on transcription factor expression) from time course data on 56 transcript expression levels obtained following different external cues. The inherently under-determined nature of this problem was addressed in part by excluding putative regulatory motifs that did not appear to be functional in multiple independent experiments from different independent external perturbations. Data were obtained from a previously published experiment in which the 56 transcripts were assayed by bioluminescence in live cells cultured on substrates of varying levels of stiffness and exposed to different levels of arginylglycylaspartic acid (RGD) peptide. The inferred dynamical networks were validated via comparison of predictions to a priori known interactions from gene databases. We discovered that exposures to different substrate stiffnesses and to RGD stimulate responses that are mediated through GATA4, SMAD3/4, ETS-1, and STAT5 and other genes, which can initiate hypertrophic, fibrotic, and inflammatory responses. The developed dynamical system identification method for discovering new mechanotransduction pathways is applicable to the identification of gene regulatory networks in numerous emerging applications where time-series data on multiple state variables and from multiple external perturbations are available.

Gene delivery from biomaterial scaffolds has been employed to induce the expression of tissue inductive factors for applications in regenerative medicine. The delivery of viral vectors has been described as reflecting a balance between vector retention and release. Herein, we investigated the design of hydrogels in order to retain the vector at the material in order to enhance transgene expression. Poly(ethylene-glycol) (PEG) hydrogels were modified with poly-l-lysine (PLL) to non-covalently bind lentivirus. For cells cultured on the hydrogels, increasing the PLL molecular weight from 1 to 70 kDa led to increased transgene expression. The incubation time of the virus with the hydrogel and the PLL concentration modulated the extent of virus adsorption, and adsorbed virus had a 20% increase in the half-life at 37°C. Alternatives to high molecular weight PLL were identified through phage display technology, with peptide sequences specific for the VSV-G ectodomain, an envelope protein pseudotyped on the virus. These affinity peptides could easily be incorporated into the hydrogel, and expression was increased 20-fold relative to control peptide, and comparable to levels observed with the high molecular weight PLL. The modification of hydrogels with affinity proteins or peptides to bind lentivirus can be a powerful strategy to enhance and localized transgene expression. Biotechnol. Bioeng. 2016;113: 2033-2040. © 2016 Wiley Periodicals, Inc.

More than half of the primordial follicles that are formed by Day 6 of postnatal life in the mouse will be eliminated from the ovary by the time of puberty. Apoptosis, a form of programmed cell death, is one mechanism by which these follicles could be actively lost. To investigate whether apoptosis is responsible for the loss of primordial follicles, follicular atresia was examined during the prepubertal period, when follicles die and are cleared from the ovary at an extremely high rate. Four hallmarks of classical apoptosis were measured in follicles present in prepubertal ovaries. The primordial follicle cohort was not positively associated with nuclear condensation or cell shrinkage, activation of caspase 3, cleavage of poly(ADP ribose) polymerase 1 (PARP1), or fragmentation of DNA. These data are consistent with a nonapoptotic pathway that is responsible for small follicle death.

Current immunotherapy for Th2-associated allergic disease is inefficient and has a significant risk of inducing anaphylaxis. There is significant need for new treatments to safely, efficiently induce antigen-specific tolerance in allergic disease. Antigen-coupled cells (Ag-SP) have been used by our lab to prevent and treat Th1/Th17-mediated models of autoimmune disease and, more recently, Th2-mediated models of allergic asthma and food allergy. Ag-SP treatment is currently entering a Phase II clinical trial in multiple sclerosis patients in Germany. Due to the technical complications of such treatment, however, we have begun to explore new antigen carriers for the induction of tolerance in autoimmune and allergic diseases. Initial work with antigen coupled to inert polystyrene and biodegradable PLGA microparticles (Ag-MP) identified these carriers as effective at inducing tolerance in EAE. We have extended these findings to a common ovalbumin (OVA)-alum induced mouse model of allergic asthma. Prophylactically, OVA- MP induce protective tolerance - inhibiting production of OVA-specific IgE antibodies and lung inflammation and preventing OVA-specific proliferation and Th2 cytokine production by restimulated lymphocytes. Given post-sensitization, OVA-MP inhibit Th2 responses in restimulation cultures but do not, in the short frame examined, inhibit other parameters of disease. This work identifies Ag-MP as promising inducers of tolerance for allergic disease.