Systemic vaccination induces CD8+ T cells and remodels the tumor microenvironment.

Therapeutic cancer vaccines are designed to increase tumor-specific T cell immunity. However, suppressive mechanisms within the tumor microenvironment (TME) may limit T cell function. Here, we assessed how the route of vaccination alters intratumoral myeloid cells. Using a self-assembling nanoparticle vaccine that links tumor antigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we treated tumor-bearing mice subcutaneously (SNP-SC) or intravenously (SNP-IV). Both routes generated antigen-specific CD8+ T cells that infiltrated tumors. However, only SNP-IV mediated tumor regression, dependent on systemic type I interferon at the time of boost. Single-cell RNA-sequencing revealed that intratumoral monocytes expressing an immunoregulatory gene signature (Chil3, Anxa2, Wfdc17) were reduced after SNP-IV boost. In humans, the Chil3+ monocyte gene signature is enriched in CD16- monocytes and associated with worse outcomes. Our results show that the generation of tumor-specific CD8+ T cells combined with remodeling of the TME is a promising approach for tumor immunotherapy.

Combination of Polymeric Micelle Formulation of TGFß Receptor Inhibitors and Paclitaxel Produce Consistent Response Across Different Mouse Models of TNBC.

Triple-negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of targetable receptors and sometimes poor response to chemotherapy. The transforming growth factor-beta (TGFß) family of proteins and their receptors (TGFR) are highly expressed in TNBC and implicated in chemotherapy-induced cancer stemness. Here we evaluated combination treatments using experimental TGFR inhibitors (TGFßi), SB525334 (SB), and LY2109761 (LY) with Paclitaxel (PTX) chemotherapy. These TGFßi target TGFR-I (SB) or both TGFR-I&II (LY). Due to the poor water solubility of these drugs, we incorporated each of them in poly(2-oxazoline) (POx) high-capacity polymeric micelles (SB-POx and LY-POx). We assessed their anti-cancer effect as single agents and in combination with micellar Paclitaxel (PTX-POx) using multiple immunocompetent TNBC mouse models that mimic human subtypes (4T1, T11-Apobec and T11-UV). While either TGFßi or PTX showed a differential effect in each model as single agents, the combinations were consistently effective against all three models. Genetic profiling of the tumors revealed differences in the expression levels of genes associated with TGFß, EMT, TLR-4, and Bcl2 signaling, alluding to the susceptibility to specific gene signatures to the treatment. Taken together, our study suggests that TGFßi and PTX combination therapy using high-capacity POx micelle delivery provides a robust anti-tumor response in multiple TNBC subtype mouse models.

Intravenous heterologous prime-boost vaccination activates innate and adaptive immunity to promote tumor regression.

Therapeutic neoantigen cancer vaccines have limited clinical efficacy to date. Here, we identify a heterologous prime-boost vaccination strategy using a self-assembling peptide nanoparticle TLR-7/8 agonist (SNP) vaccine prime and a chimp adenovirus (ChAdOx1) vaccine boost that elicits potent CD8 T cells and tumor regression. ChAdOx1 administered intravenously (i.v.) had 4-fold higher antigen-specific CD8 T cell responses than mice boosted by the intramuscular (i.m.) route. In the therapeutic MC38 tumor model, i.v. heterologous prime-boost vaccination enhances regression compared with ChAdOx1 alone. Remarkably, i.v. boosting with a ChAdOx1 vector encoding an irrelevant antigen also mediates tumor regression, which is dependent on type I IFN signaling. Single-cell RNA sequencing of the tumor myeloid compartment shows that i.v. ChAdOx1 reduces the frequency of immunosuppressive Chil3 monocytes and activates cross-presenting type 1 conventional dendritic cells (cDC1s). The dual effect of i.v. ChAdOx1 vaccination enhancing CD8 T cells and modulating the TME represents a translatable paradigm for enhancing anti-tumor immunity in humans.

Preparation of an Orthotopic, Syngeneic Model of Lung Adenocarcinoma and the Testing of the Antitumor Efficacy of Poly(2-oxazoline) Formulation of Chemo-and Immunotherapeutic Agents.

Tumor xenograft models developed by transplanting human tissues or cells into immune-deficient mice are widely used to study human cancer response to drug candidates. However, immune-deficient mice are unfit for investigating the effect of immunotherapeutic agents on the host immune response to cancer (Morgan, 2012). Here, we describe the preparation of an orthotopic, syngeneic model of lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), to study the antitumor effect of chemo and immunotherapeutic agents in an immune-competent animal. The tumor model is developed by implanting 344SQ LUAD cells derived from the metastases of KrasG12D; p53R172HΔG genetically engineered mouse model into the left lung of a syngeneic host (Sv/129). The 344SQ LUAD model offers several advantages over other models: 1) The immune-competent host allows for the assessment of the biologic effects of immune-modulating agents; 2) The pathophysiological features of the human disease are preserved due to the orthotopic approach; 3) Predisposition of the tumor to metastasize facilitates the study of therapeutic effects on primary tumor as well as the metastases ( Chen et al., 2014 ). Furthermore, we also describe a treatment strategy based on Poly(2-oxazoline) micelles that has been shown to be effective in this difficult-to-treat tumor model ( Vinod et al., 2020b ).

Preparation and Characterization of Poly(2-oxazoline) Micelles for the Solubilization and Delivery of Water Insoluble Drugs.

Many new drug development candidates are highly lipophilic compounds with low water solubility. This constitutes a formidable challenge for the use of such compounds for cancer therapy, where high doses and intravenous injections are needed ( Di et al., 2012 ). Here, we present a poly(2-oxazoline) polymer (POx)-based nanoformulation strategy to solubilize and deliver hydrophobic drugs. POx micelles are prepared by a simple thin-film hydration method. In this method, the drug and polymer are dissolved in a common solvent and allowed to mix, following which the solvent is evaporated using mild heating conditions to form a thin film. The micelles form spontaneously upon hydration with saline. POx nanoformulation of hydrophobic drugs is unique in that it has a high drug loading capacity, which is superior to micelles of conventional surfactants. Moreover, multiple active pharmaceutical ingredients (APIs) can be included within the same POx micelle, thereby enabling the codelivery of binary as well as ternary drug combinations ( Han et al., 2012 ; He et al., 2016 ).

High-capacity poly(2-oxazoline) formulation of TLR 7/8 agonist extends survival in a chemo-insensitive, metastatic model of lung adenocarcinoma.

About 40% of patients with non-small cell lung cancer (NSCLC) have stage IV cancer at the time of diagnosis. The only viable treatment options for metastatic disease are systemic chemotherapy and immunotherapy. Nonetheless, chemoresistance remains a major cause of chemotherapy failure. New immunotherapeutic modalities such as anti-PD-1 immune checkpoint blockade have shown promise; however, response to such strategies is highly variable across patients. Here, we show that our unique poly(2-oxazoline)-based nanomicellar formulation (PM) of Resiquimod, an imidazoquinoline Toll-like receptor (TLR) 7/8 agonist, had a superior tumor inhibitory effect in a metastatic model of lung adenocarcinoma, relative to anti-PD-1 therapy or platinum-based chemotherapy. Investigation of the in vivo immune status following Resiquimod PM treatment showed that Resiquimod-based stimulation of antigen-presenting cells in the tumor microenvironment resulted in the mobilization of an antitumor CD8+ immune response. Our study demonstrates the promise of poly(2-oxazoline)-formulated Resiquimod for treating metastatic NSCLC.

A high proportion of red snapper sold in North Carolina is mislabeled.

Seafood mislabeling occurs when a market label is inaccurate, primarily in terms of species identity, but also regarding weight, geographic origin, or other characteristics. This widespread problem allows cheaper or illegally-caught species to be marketed as species desirable to consumers. Previous studies have identified red snapper (Lutjanus campechanus) as one of the most frequently mislabeled seafood species in the United States. To quantify how common mislabeling of red snapper is across North Carolina, the Seafood Forensics class at the University of North Carolina at Chapel Hill used DNA barcoding to analyze samples sold as "red snapper" from restaurants, seafood markets, and grocery stores purchased in ten counties. Of 43 samples successfully sequenced and identified, 90.7% were mislabeled. Only one grocery store chain (of four chains tested) accurately labeled red snapper. The mislabeling rate for restaurants and seafood markets was 100%. Vermilion snapper (Rhomboplites aurorubens) and tilapia (Oreochromis aureus and O. niloticus) were the species most frequently substituted for red snapper (13 of 39 mislabeled samples for both taxa, or 26 of 39 mislabeled total). This study builds on previous mislabeling research by collecting samples of a specific species in a confined geographic region, allowing local vendors and policy makers to better understand the scope of red snapper mislabeling in North Carolina. This methodology is also a model for other academic institutions to engage undergraduate researchers in mislabeling data collection, sample processing, and analysis.