Biomaterial-Mediated Genetic Reprogramming of Merkel Cell Carcinoma and Melanoma Leads to Targeted Cancer Cell Killing In Vitro and In Vivo.

Tumor immunotherapy is a promising anticancer strategy; however, tumor cells may employ resistance mechanisms, including downregulation of major histocompatibility complex (MHC) molecules to avoid immune recognition. Here, we investigate reprogramming nanoparticles (NPs) that deliver immunostimulatory genes to enhance immunotherapy and address defective antigen presentation in skin cancer in vitro and in vivo. We use a modular poly(beta-amino ester) (PBAE)-based NP to deliver DNA encoding 4-1BBL, IL-12, and IFNγ to reprogram human Merkel cell carcinoma (MCC) cells in vitro and mouse melanoma tumors in vivo to drive adaptive antitumor immune responses. Optimized NP formulations delivering 4-1BBL/IL-12 or 4-1BBL/IL-12/IFNγ DNA successfully transfect MCC and melanoma cells in vitro and in vivo, respectively, resulting in IFNγ-driven upregulation of MHC class I and II molecules on cancer cells. These NPs reprogram the tumor immune microenvironment (TIME) and elicit strong T-cell-driven immune responses, leading to cancer cell killing and T-cell proliferation in vitro and slowing tumor growth and improving survival rates in vivo. Based on expected changes to the tumor immune microenvironment, particularly the importance of IFNγ to the immune response and driving both T-cell function and exhaustion, next-generation NPs codelivering IFNγ were designed. These offered mixed benefits, exchanging improved polyfunctionality for increased T-cell exhaustion and demonstrating higher systemic toxicity in vivo. Further profiling of the immune response with these NPs provides insight into T-cell exhaustion and polyfunctionality induced by different formulations, providing a greater understanding of this immunotherapeutic strategy.

Pembrolizumab-Induced Lichen Planus Pemphigoides in a Patient with Metastatic Adrenocortical Cancer: A Case Report and Literature Review.

While the advent of immune-checkpoint inhibitors has revolutionized cancer therapy, immune-related adverse effects (irAEs) have also been on the rise. Cutaneous toxicities are among the most common irAEs, especially in the context of programmed cell death protein-1 (PD-1) inhibitors like pembrolizumab. Herein, we report a case of anti-PD-1-induced lichen planus pemphigoides (LPP)-a rare autoimmune blistering disorder with characteristics of both lichen planus and bullous pemphigoid. To our knowledge, this is the first reported case of LPP following anti-PD-1 therapy for metastatic adrenocortical cancer. Recognizing that LPP is within the spectrum of irAEs is important, especially as the indications for immunotherapy grow to include rarer malignancies like adrenocortical cancer. In addition to our case presentation, we also provide a comprehensive review of the literature surrounding immunotherapy-induced LPP-highlighting key characteristics towards the early recognition and clinical management of this cutaneous irAE.

Whole-Slide Imaging, Mutual Information Registration for Multiplex Immunohistochemistry and Immunofluorescence.

Multiplex immunohistochemistry/immunofluorescence (mIHC/mIF) is a developing technology that facilitates the evaluation of multiple, simultaneous protein expressions at single-cell resolution while preserving tissue architecture. These approaches have shown great potential for biomarker discovery, yet many challenges remain. Importantly, streamlined cross-registration of multiplex immunofluorescence images with additional imaging modalities and immunohistochemistry (IHC) can help increase the plex and/or improve the quality of the data generated by potentiating downstream processes such as cell segmentation. To address this problem, a fully automated process was designed to perform a hierarchical, parallelizable, and deformable registration of multiplexed digital whole-slide images (WSIs). We generalized the calculation of mutual information as a registration criterion to an arbitrary number of dimensions, making it well suited for multiplexed imaging. We also used the self-information of a given IF channel as a criterion to select the optimal channels to use for registration. Additionally, as precise labeling of cellular membranes in situ is essential for robust cell segmentation, a pan-membrane immunohistochemical staining method was developed for incorporation into mIF panels or for use as an IHC followed by cross-registration. In this study, we demonstrate this process by registering whole-slide 6-plex/7-color mIF images with whole-slide brightfield mIHC images, including a CD3 and a pan-membrane stain. Our algorithm, WSI, mutual information registration (WSIMIR), performed highly accurate registration allowing the retrospective generation of an 8-plex/9-color, WSI, and outperformed 2 alternative automated methods for cross-registration by Jaccard index and Dice similarity coefficient (WSIMIR vs automated WARPY, P < .01 and P < .01, respectively, vs HALO + transformix, P = .083 and P = .049, respectively). Furthermore, the addition of a pan-membrane IHC stain cross-registered to an mIF panel facilitated improved automated cell segmentation across mIF WSIs, as measured by significantly increased correct detections, Jaccard index (0.78 vs 0.65), and Dice similarity coefficient (0.88 vs 0.79).

Disseminated Tuberculosis With an Atypical Cutaneous Manifestation in a Hematopoietic Cell Transplant Patient in the Early Posttransplant Period: Case Report and Review of the Literature.

We describe an unusual case of posttransplant tuberculosis reactivation in a man who underwent allogeneic hematopoietic cell transplant. Concomitant with disseminated adenovirus infection, reactivation of tuberculosis manifested as disseminated, nonfollicular pustules on day +49. Skin biopsy was obtained on day +50. Initial histopathologic evaluation did not suggest mycobacterial infection, but tissue stain showed acid-fast organisms, which were subsequently identified as Mycobacterium tuberculosis. Shortly after the cutaneous presentation of tuberculosis, the patient died on day +52. Our case is among a paucity of reports describing tuberculosis reactivation in hematopoietic cell transplant patients in the early posttransplant period. It highlights the difficulty of diagnosing contemporaneous systemic infections, and it presents a rare and atypical cutaneous manifestation of tuberculosis in a hematopoietic cell transplant patient. Our case and review of the literature emphasize the need for further research to elucidate risk factors associated with early posttransplant reactivation of tuberculosis, and the importance of remaining vigilant for active tuberculosis in hematopoietic cell transplant patients with epidemiologic risk factors.

Immune cell subsets in interface cutaneous immune-related adverse events associated with anti-PD-1 therapy resemble acute graft versus host disease more than lichen planus.

BACKGROUND: Checkpoint immunotherapy is frequently associated with cutaneous immune-related adverse events (cirAEs), and among those, the most common subtype shows interface reaction patterns that have been likened to lichen planus (LP); however, cutaneous acute graft versus host disease (aGVHD) may be a closer histopathologic comparator. We used quantitative pathology to compare the immunologic composition of anti-PD-1-associated interface reactions to LP and aGVHD to assess for similarities and differences between these cutaneous eruptions. METHODS: Immunohistochemistry for CD4, CD8, CD68, PD-1, and PD-L1 was performed on formalin-fixed paraffin-embedded tissue from patients with anti-PD-1 interface cirAEs (n = 4), LP (n = 9), or aGVHD (n = 5). Densities of immune cell subsets expressing each marker were quantified using the HALO image analysis immune cell module. Plasma cell and eosinophil density were quantified on routine H&E slides. RESULTS: Specimens from patients with anti-PD-1 interface cirAEs showed equivalent total cell densities and immune cell composition to those with aGVHD. Patients with LP showed higher total immune cell infiltration, higher absolute T-cell densities, increased CD8 proportion, and reduced histiocytic component. The cases with the highest plasma cell counts were all anti-PD-1 interface cirAEs and aGVHD. CONCLUSION: The composition of immune cell subsets in anti-PD-1 interface cirAEs more closely resembles the immune response seen in aGVHD than LP within our cohort. This warrants a closer look via advanced analytics and may have implications for shared pathogenesis and potential treatment options.

Actinic Keratosis Color and Its Associations: A Retrospective Photographic, Dermoscopic, and Histologic Evaluation.

BACKGROUND AND OBJECTIVE: Actinic keratoses (AKs) have been described with varying color and morphology; however, no reports have demonstrated associations between color, vasculature, and inflammation. In this retrospective study, we analyze the clinical, dermoscopic, and histopathologic features of AKs to elucidate this relationship. METHODS: A retrospective search for patients diagnosed with AK between January 2018 and October 2019 was performed. Clinical and dermoscopic photographs and pathology slides for all included subjects were reviewed. RESULTS: Forty-nine images and histopathology slides were analyzed. Dermoscopy of white AKs demonstrated scale and absence of erythema with corresponding absence of inflammation on histopathology. Dermoscopy of brown AKs revealed pseudonetwork, absent scale, and a variable vessel pattern with pigment incontinence and absence of inflammation on histopathology. Red AKs had a distinct polymorphous vessel pattern and presence of erythema on dermoscopy. On histopathology, about half of samples showed increased vascularity and variable inflammation. Pink AK dermoscopy revealed a presence of erythema with corresponding presence of inflammation on histopathology. CONCLUSION: This report adds to our understanding of AKs and confirms that, in general, the pinker or redder the AK, the more prominent the inflammatory infiltrate and vasculature, respectively. Dermatologists should continue to use their diagnostic skills to successfully diagnose and triage AKs.

IFNα and 5-Aza-2'-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model.

Introduction: DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival. Methods: Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC). Results: The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy. Discussion: Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.

High-throughput evaluation of polymeric nanoparticles for tissue-targeted gene expression using barcoded plasmid DNA.

Successful systemic gene delivery requires specific tissue targeting as well as efficient intracellular transfection. Increasingly, research laboratories are fabricating libraries of novel nanoparticles, engineering both new biomaterial structures and composition ratios of multicomponent systems. Yet, methods for screening gene delivery vehicles directly in vivo are often low-throughout, limiting the number of candidate nanoparticles that can be investigated. Here, we report a comprehensive, high-throughput method to evaluate a library of polymeric nanoparticles in vivo for tissue-specific gene delivery. The method involves pairing each nanoparticle formulation with a plasmid DNA (pDNA) that harbors a unique nucleotide sequence serving as the identifying "barcode". Using real time quantitative PCR (qPCR) for detection of the barcoded pDNA and quantitative reverse transcription PCR (RT-qPCR) for transcribed barcoded mRNA, we can quantify accumulation and transfection in tissues of interest. The barcode pDNA and primers were designed with sufficient sensitivity and specificity to evaluate multiple nanoparticle formulations per mouse, improving screening efficiency. Using this platform, we evaluated the biodistribution and transfection of 8 intravenously administered poly(beta-amino ester; PBAE) nanoparticle formulations, each with a PBAE polymer of differential structure. Significant levels of nanoparticle accumulation and gene transfection were observed mainly in organs involved in clearance, including spleen, liver, and kidneys. Interestingly, higher levels of transfection of select organs did not necessarily correlate with higher levels of tissue accumulation, highlighting the importance of directly measuring in vivo transfection efficiency as the key barcoded parameter in gene delivery vector optimization. To validate this method, nanoparticle formulations were used individually for luciferase pDNA delivery in vivo. The distribution of luciferase expression in tissues matched the transfection analysis by the barcode qPCR method, confirming that this platform can be used to accurately evaluate systemic gene delivery.

Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD-1 blockade.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163+PD-L1- myeloid cells and CD8+FoxP3+PD-1low/mid T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution.

A Series of RET Fusion Spitz Neoplasms With Plaque-Like Silhouette and Dyscohesive Nesting of Epithelioid Melanocytes.

ABSTRACT: Two distinct studies have shown that RET fusions are found in 3%-4% of Spitz neoplasms. RET fusions have been well described in papillary thyroid cancer, non-small-cell lung cancer, breast cancer, and soft-tissue mesenchymal tumors as well as some other neoplasms. However, there are no comprehensive descriptions to date of the characteristic morphologic, clinical, or genomic findings in RET fusion Spitz neoplasms. In this study, we identified 5 cases of RET fusion Spitz neoplasms. These tumors showed characteristic morphologic features which included plaque-like silhouette and monotonous epithelioid cytology with expansile and dyscohesive nesting. Four of 5 patients including 1 diagnosed as Spitz melanoma had clinical follow-up all of which was uneventful. Furthermore, we describe the genomic sequences in 4 of these cases, 2 of which have previously described KIF5B-RET fusion and 2 of which had a novel LMNA-RET fusion. We believe this report significantly contributes to our current knowledge regarding Spitz neoplasms and describes characteristics features which can help with recognition of the RET subgroup of Spitz.

Clinical, morphologic, and genomic findings in ROS1 fusion Spitz neoplasms.

The presence of a characteristic chimeric fusion as the initiating genomic event is one defining feature of Spitz neoplasms. Characterization of specific subtypes of Spitz neoplasms allows for better recognition facilitating diagnosis. Data on clinical outcomes of the specific tumor types may help in predicting behavior. In this study we present the largest series to date on ROS1 fusion Spitz neoplasms. We present the clinical, morphologic, and genomic features of 17 cases. We compared the morphologic features of these 17 cases to a cohort of 99 other non-ROS1 Spitz neoplasms to assess for features that may have high specificity for ROS1 fusions. These tumors consisted of ten Spitz nevi and seven Spitz tumors. None of the cases met criteria for a diagnosis of Spitz melanoma. Morphologically, the ROS1 fusion tumors of this series were characterized by a plaque-like or nodular silhouette, often densely cellular intraepidermal melanocyte proliferation, frequent pagetosis, tendency toward spindle cell cytomorphology, low grade nuclear atypia, and floating nests with occasional transepidermal elimination. However, there was a significant range in microscopic appearances, including two cases with morphologic features of a desmoplastic Spitz nevus. Different binding partners to ROS1 were identified with PWWP2A and TPM3 being the most common. No case had a recurrence or metastasis. Our findings document that most ROS1 fusion Spitz neoplasms have some typical characteristic microscopic features, while a small proportion will have features overlapping with other genomic subtypes of Spitz neoplasms. Preliminary evidence suggests that they tend to be indolent or low grade neoplasms.

Melanocytic Neoplasms With MAP2K1 in Frame Deletions and Spitz Morphology.

With the advent of better molecular characterization of Spitz melanocytic neoplasms, there has been increasing effort to better understand and describe the relationships between specific driver fusion and/or mutations with the clinical and histomorphological characteristics of the lesions. Structural rearrangements in mitogen activated protein kinase genes have recently been noted to be important in Spitz neoplasms. Only very few reports, however, have described in detail melanocytic tumors with in frame deletions in MAP2K1. Cases in the literature with this aberration have been described as having a diagnosis of Spitz, deep penetrating nevi, or pigmented epithelioid melanocytoma. In this study, we describe a cohort of 6 cases with MAP2K1 activating in frame deletions. The morphologic spectrum of the cases was broad. Common features of these cases include Spitzoid cytomorphology (5/6) cases, prominent melanin pigmentation (4/6) cases, and deep penetrating nevi-like plexiform architecture (3/6) cases. The diagnoses at the time of clinical care of these cases included nevus of Reed (1/6), desmoplastic Spitz tumor (1/6), BAPoma (1/6), deep penetrating melanocytic nevus (2/6), and melanoma (1/6). Clinical follow-up was available in 3 of the 6 cases. None of the patients had a tumor recurrence. This builds on the growing literature to help expand the spectrum of changes associated with Spitzoid melanocytic neoplasms.

BRAF fusion Spitz neoplasms; clinical morphological, and genomic findings in six cases.

BACKGROUND: Fusions involving the BRAF gene are responsible for 5% of Spitz neoplasms. To better characterize them, we report the clinical, morphological, and genomic findings of six BRAF fusion Spitz tumors. METHODS: The morphological, clinical, and molecular findings of six BRAF fusion Spitz neoplasms assessed by next generation sequencing (NGS) were compared to a control set of Spitz without BRAF fusions. RESULTS: BRAF fusion Spitz tumors had frequent predominance of epithelioid morphology (4/6 cases), frequent high-grade nuclear atypia and pleomorphism (5/6 cases), and a frequent desmoplastic base (3/6 cases). Five of six cases were diagnosed as atypical Spitz tumor and one as Spitz nevus. All cases had uneventful clinical follow-up. There were five different fusion partners, with CLIP2 being the most frequent. Secondary pathogenic mutations were frequent and chromosomal copy number changes were seen in three of six cases by an NGS platform. CONCLUSIONS: BRAF fusions Spitz usually have epithelioid morphology, high-grade nuclear atypia, and desmoplasia. Chromosomal copy number changes are not infrequent. While our cases had uneventful follow-up, a meta-analysis of the literature suggests that among the fusion subtypes associated with Spitz tumors, they are among the subgroups more likely to develop distant metastasis.

Successful Treatment of In-Transit Metastatic Melanoma in a Renal Transplant Patient With Combination T-VEC/Imiquimod Immunotherapy.

In the era of immunotherapy for cancer, solid organ transplant patients who go on to develop metastatic or locally advanced melanoma offer particularly difficult challenges. New approaches are needed for these patients. We present a case of in-transit metastatic melanoma in a renal transplant patient. The patient was initially managed with talimogene laherparepvec (T-VEC) injections alone with continued local progression. Addition of topical imiquimod 5% cream to intralesional T-VEC resulted in a rapid and dramatic response, with complete clearance of the cutaneous in-transit metastases and without any sign of organ rejection. In solid organ transplant patients who lack surgical options and are not eligible for treatment with a BRAF inhibitor, and for whom treatment with checkpoint inhibitors present risk of organ rejection, T-VEC either alone or in combination with topical imiquimod should be considered for patients with locally advanced disease. This combination should be a consideration, with close observation, in patients with a history of organ transplantation and immunosuppression.

Which Nanobasics Should Be Taught in Medical Schools?

The progressive growth in nanotechnology approaches to diagnostics and therapeutics, especially for cancer, necessitates training physicians in nanoethics. This article explains why it is critical for medical education to include instruction in nanotechnology, nanomedicine, nanotoxicology, and nanoethics and suggests basic concepts educators can use to infuse curricula with this content.

Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab.

BACKGROUND: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. METHODS: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. RESULTS: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm2, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 µm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. CONCLUSIONS: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, Tregs, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.

Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.

There has been growing interest in the use of particles coated with lipids for applications ranging from drug delivery, gene delivery, and diagnostic imaging to immunoengineering. To date, almost all particles with lipid coatings have been spherical despite emerging evidence that non-spherical shapes can provide important advantages including reduced non-specific elimination and increased target-specific binding. We combine control of core particle geometry with control of particle surface functionality by developing anisotropic, biodegradable ellipsoidal particles with lipid coatings. We demonstrate that these lipid coated ellipsoidal particles maintain advantageous properties of lipid polymer hybrid particles, such as the ability for modular protein conjugation to the particle surface using versatile bioorthogonal ligation reactions. In addition, they exhibit biomimetic membrane fluidity and demonstrate lateral diffusive properties characteristic of natural membrane proteins. These ellipsoidal particles simultaneously provide benefits of non-spherical particles in terms of stability and resistance to non-specific phagocytosis by macrophages as well as enhanced targeted binding. These biomaterials provide a novel and flexible platform for numerous biomedical applications. STATEMENT OF SIGNIFICANCE: The research reported here documents the ability of non-spherical polymeric particles to be coated with lipids to form anisotropic biomimetic particles. In addition, we demonstrate that these lipid-coated biodegradable polymeric particles can be conjugated to a wide variety of biological molecules in a "click-like" fashion. This is of interest due to the multiple types of cellular mimicry enabled by this biomaterial based technology. These features include mimicry of the highly anisotropic shape exhibited by cells, surface presentation of membrane bound protein mimetics, and lateral diffusivity of membrane bound substrates comparable to that of a plasma membrane. This platform is demonstrated to facilitate targeted cell binding while being resistant to non-specific cellular uptake. Such a platform could allow for investigations into how physical parameters of a particle and its surface affect the interface between biomaterials and cells, as well as provide biomimetic technology platforms for drug delivery and cellular engineering.

Biodegradable STING agonist nanoparticles for enhanced cancer immunotherapy.

Therapeutic cancer vaccines require adjuvants leading to robust type I interferon and proinflammatory cytokine responses in the tumor microenvironment to induce an anti-tumor response. Cyclic dinucleotides (CDNs), a potent Stimulator of Interferon Receptor (STING) agonist, are currently in phase I trials. However, their efficacy may be limited to micromolar concentrations due to the cytosolic residence of STING in the ER membrane. Here we utilized biodegradable, poly(beta-amino ester) (PBAE) nanoparticles to deliver CDNs to the cytosol leading to robust immune response at >100-fold lower extracellular CDN concentrations in vitro. The leading CDN PBAE nanoparticle formulation induced a log-fold improvement in potency in treating established B16 melanoma tumors in vivo when combined with PD-1 blocking antibody in comparison to free CDN without nanoparticles. This nanoparticle-mediated cytosolic delivery method for STING agonists synergizes with checkpoint inhibitors and has strong potential for enhanced cancer immunotherapy.