Myeloid cell reprogramming alleviates immunosuppression and promotes clearance of metastatic lesions.

Suppressive myeloid cells, including monocyte and neutrophil populations, play a vital role in the metastatic cascade and can inhibit the anti-tumor function of cytotoxic T-cells. Cargo-free polymeric nanoparticles (NPs) have been shown to modulate innate immune cell responses in multiple pathologies of aberrant inflammation. Here, we test the hypothesis that the intravenous administration of drug-free NPs in the 4T1 murine model of metastatic triple-negative breast cancer can reduce metastatic colonization of the lungs, the primary metastatic site, by targeting the pro-tumor immune cell mediators of metastatic progression. In vivo studies demonstrated that NP administration reprograms the immune milieu of the lungs and reduces pulmonary metastases. Single-cell RNA sequencing of the lungs revealed that intravenous NP administration alters myeloid cell phenotype and function, skewing populations toward inflammatory, anti-tumor phenotypes and away from pro-tumor phenotypes. Monocytes, neutrophils, and dendritic cells in the lungs of NP-treated mice upregulate gene pathways associated with IFN signaling, TNF signaling, and antigen presentation. In a T-cell deficient model, NP administration failed to abrogate pulmonary metastases, implicating the vital role of T-cells in the NP-mediated reduction of metastases. NPs delivered as an adjuvant therapy, following surgical resection of the primary tumor, led to clearance of established pulmonary metastases in all treated mice. Collectively, these results demonstrate that the in vivo administration of cargo-free NPs reprograms myeloid cell responses at the lungs and promotes the clearance of pulmonary metastases in a method of action dependent on functional T-cells.

Implications of TGFß Signaling and CDK Inhibition for the Treatment of Breast Cancer.

TGFß signaling enacts tumor-suppressive functions in normal cells through promotion of several cell regulatory actions including cell-cycle control and apoptosis. Canonical TGFß signaling proceeds through phosphorylation of the transcription factor, SMAD3, at the C-terminus of the protein. During oncogenic progression, this tumor suppressant phosphorylation of SMAD3 can be inhibited. Overexpression of cyclins D and E, and subsequent hyperactivation of cyclin-dependent kinases 2/4 (CDKs), are often observed in breast cancer, and have been associated with poor prognosis. The noncanonical phosphorylation of SMAD3 by CDKs 2 and 4 leads to the inhibition of tumor-suppressive function of SMAD3. As a result, CDK overactivation drives oncogenic progression, and can be targeted to improve clinical outcomes. This review focuses on breast cancer, and highlights advances in the understanding of CDK-mediated noncanonical SMAD3 phosphorylation. Specifically, the role of aberrant TGFß signaling in oncogenic progression and treatment response will be examined to illustrate the potential for therapeutic discovery in the context of cyclins/CDKs and SMAD3.

Disease-induced immunomodulation at biomaterial scaffolds detects early pancreatic cancer in a spontaneous model.

Pancreatic cancer has the worst prognosis of all cancers due to disease aggressiveness and paucity of early detection platforms. We developed biomaterial scaffolds that recruit metastatic tumor cells and reflect the immune dysregulation of native metastatic sites. While this platform has shown promise in orthotopic breast cancer models, its potential in other models is untested. Herein, we demonstrate that scaffolds recruit disseminated pancreatic cells in the KPCY model of spontaneous pancreatic cancer prior to adenocarcinoma formation (3-fold increase in scaffold YFP + cells). Furthermore, immune cells at the scaffolds differentiate early- and late-stage disease with greater accuracy (0.83) than the natural metastatic site (liver, 0.50). Early disease was identified by an approximately 2-fold increase in monocytes. Late-stage disease was marked by a 1.5-2-fold increase in T cells and natural killer cells. The differential immune response indicated that the scaffolds could distinguish spontaneous pancreatic cancer from spontaneous breast cancer. Collectively, our findings demonstrate the utility of scaffolds to reflect immunomodulation in two spontaneous models of tumorigenesis, and their particular utility for identifying early disease stages in the aggressive KPCY pancreatic cancer model. Such scaffolds may serve as a platform for early detection of pancreatic cancer to improve treatment and prognosis.

Cyclin E overexpression confers resistance to trastuzumab through noncanonical phosphorylation of SMAD3 in HER2+ breast cancer.

The efficacy of trastuzumab, a treatment for HER2+ breast cancer, can be limited by the development of resistance. Cyclin E (CCNE) overexpression has been implicated in trastuzumab resistance. We sought to uncover a potential mechanism for this trastuzumab resistance and focused on a model of CCNE overexpressing HER2+ breast cancer and noncanonical phosphorylation of the TGF-ß signaling protein, SMAD3. Network analysis of transcriptional activity in a HER2+, CCNE overexpressing, trastuzumab-resistant cell line (BT474R2) identified decreased SMAD3 activity was associated with treatment resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p < .01), and noncanonical phosphorylation of SMAD3 was increased in these CCNE-overexpressing cells. Also, in response to CDK2 inhibition, expression patterns linked to restored canonical SMAD3 signaling, including decreased cMyc and increased cyclin-dependent inhibitor, p15, were identified. The BT474R2 cell line was modified through overexpression of SMAD3 (BT474R2-SMAD3), a mutant construct resistant to CCNE-mediated noncanonical phosphorylation of SMAD3 (BT474R2-5M), and a control (BT474R2-Blank). In vitro studies examining the response to trastuzumab showed increased sensitivity to treatment for BT474R2-5M cells. These findings were then validated in NSG mice inoculated with BT474R2-5M cells or BT474R2 control cells. After treatment with trastuzumab, the NSG mice inoculated with BT474R2-5M cells developed significantly lower tumor volumes (p < .001), when compared to mice inoculated with BT474R2 cells. Taken together, these results indicate that for patients with HER2+ breast cancer, a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, could be treated with CDK2 inhibition to help enhance the efficacy of trastuzumab therapy.

Combinatorial, Microparticle-Based Delivery of Immune Modulators Reprograms the Dendritic Cell Phenotype and Promotes Remission of Collagen-Induced Arthritis in Mice.

Safe, effective, antigen-specific therapy for rheumatoid arthritis (RA) remains an elusive clinical goal with a few lasting, viable options on the horizon. Existing therapeutic interventions are indiscriminate and inconsistently immunosuppressive, often leaving patients susceptible to infection. Herein, we investigate the use of a dual-sized, microparticle "regulatory vaccine" (REGvac) that passively targets dendritic cells for antigen-specific biomaterial-based immunotherapy of RA. This REGvac employs poly(d,l-lactic-co-glycolic-acid) (PLGA) microparticles (MPs) encapsulating (i) a dendritic cell chemoattractant, (ii) potent immunosuppressive molecules, (iii) and an RA-relevant autoantigen to provide a multifaceted approach for the treatment of collagen-induced arthritis (CIA), the primary mouse model of RA. Subcutaneous administrations of the REGvac after mice had developed moderate clinical symptoms markedly diminished overt inflammation in the paws, halted cartilage degradation, and restored gait parameters within 56 days after initial treatment. Positron emission tomography imaging corroborated reduction of inflammation in the paws of REGvac-treated mice. In-depth immunological assessments showed a decreased expression of CD80, CD86, and MHC II on CD11c+ dendritic cells in joint-associated lymph nodes. Further, we observed significant increases in conventional regulatory CD25+FOXP3+ T cells, as well as programmed cell death protein-1 (PD-1)-expressing CD4+ T cells in joint-proximal lymph nodes and the spleen. Real-time PCR analysis of joint tissues from treated mice revealed significant decreases in inflammatory cytokine expression (IL-6), while IL-10 mRNA levels were significantly increased. These observations strongly hint toward the induction of multiple tolerogenic mechanisms by administration of this MP regulatory vaccine. With regards to antigen specificity, ex vivo antigen recall assays revealed a lack of response to collagen by CD4+ T cells from the popliteal and inguinal lymph nodes of REGvac-treated mice, contrasting with the proliferative response of CD4+ T cells from CIA+ mice. Taken altogether, our results strongly support the application of this MP regulatory vaccine as a potent, biomaterial-based, antigen-specific therapy for RA.

Latent, Immunosuppressive Nature of Poly(lactic-co-glycolic acid) Microparticles.

Use of biomaterials to spatiotemporally control the activation of immune cells is at the forefront of biomedical engineering research. As more biomaterial strategies are employed for immunomodulation, understanding the immunogenicity of biodegradable materials and their byproducts is paramount in tailoring systems for immune activation or suppression. Poly(D,L-lactic-co-glycolic acid) (PLGA), one of the most commonly studied polymers in tissue engineering and drug delivery, has been previously described on one hand as an immune adjuvant, and on the other as a nonactivating material. In this study, the effect of PLGA microparticles (MPs) on the maturation status of murine bone marrow-derived dendritic cells (DCs), the primary initiators of adaptive immunity, was investigated to decipher the immunomodulatory properties of this biomaterial. Treatment of bone marrow-derived DCs from C57BL/6 mice with PLGA MPs led to a time dependent decrease in the maturation level of these cells, as quantified by decreased expression of the positive stimulatory molecules MHCII, CD80, and CD86 as well as the ability to resist maturation following challenge with lipopolysaccharide (LPS). Moreover, this immunosuppression was dependent on the molecular weight of the PLGA used to fabricate the MPs, as higher molecular weight polymers required longer incubation to produce comparable dampening of maturation molecules. These phenomena were correlated to an increase in lactic acid both intracellularly and extracellularly during DC/PLGA MP coculture, which is postulated to be the primary agent behind the observed immune inhibition. This hypothesis is supported by our results demonstrating that resistance to LPS stimulation may be due to the ability of PLGA MP-derived lactic acid to inhibit the phosphorylation of TAK1 and therefore prevent NF-κB activation. This work is significant as it begins to elucidate how PLGA, a prominent biomaterial with broad applications ranging from tissue engineering to pharmaceutics, could modulate the local immune environment and offers insight on engineering PLGA to exploit its evolving immunogenicity.