Markovic, Svetomir N.

Abstract The sentinel lymph node (SLN) is often the first site of melanoma spread and a critical immune checkpoint that determines local tumor dissemination to systemic metastasis. Although lymph nodes are central to antitumor immunity, the mechanisms that render them permissive to metastasis remain poorly understood. This study used spatial imaging to characterize immune and stromal phenotypic alteration in tumor-negative SLNs (SLN-) and tumor-positive SLNs (SLN+) from patients with cutaneous melanoma, compared to non-melanoma-associated normal lymph nodes (NLN). We performed multiplex immunofluorescence (MxIF) analysis of formalin-fixed, paraffin-embedded lymph node tissues from patients with cutaneous melanoma and control subjects, including two subjects per group (NLN, SLN-, and SLN+). Furthermore, quantitative single-cell segmentation and classification using QuPath generated median fluorescence intensities for immune, stromal, and tumor markers across LN, SLN- and SLN+ regions of interest. In addition to the initial analysis, we employed spatial neighborhood analysis to characterize markers expression profiles surrounding target immune population. This approach quantified local microenvironment based on marker normalized intensity-based co-expression pattern and stromal remodeling signatures. We grouped these markers based on the functional panels representing T-cell activation and exhaustion, myeloid polarization, tumor interaction, stromal remodeling, and apoptosis pathways providing insights into how prometastatic niche evolve within SLN. Normalized data, via centered log ratio (CLR) transformation and Gaussian Mixture Model (GMM) gating was analyzed using principal component analysis, UMAP/tSNE embedding, and hierarchical clustering in in python. Progressive immune remodeling was observed from normal NLN to SLN+, including expansion of regulatory T cells, enrichment of exhausted T cells as well as myeloid-derived suppressor cells (MDSC), and transition toward an M2-like macrophage phenotype. The spatial data from SLN- samples showed an early sign of immune suppression, with reduced cytotoxic capacity. These results show that SLN gets features of peripheral immune tolerance for melanoma early, prior to pathological dissection of metastatic spread supporting the existence of a pre-metastatic niche that facilitates lymphatic colonization of tumor cells. This work provides insights into early immune escape and may inform strategies for immunotherapy and early intervention in melanoma. Citation Format: Shankar Suman, Wendy K. Nevala, Noah A. Stueven, Raymond M. Moore, Chathu L. Atherton, Jeffrey E. Johnson, Ray Guo, James W. Jakub, Svetomir N. Markovic. Spatial remodeling of immune and stromal microenvironments in sentinel lymph nodes during melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6193.

Abstract Introduction: Placental cell-free DNA (cfDNA) activates maternal monocytes to a proinflammatory phenotype through cGAS-STING signaling during pregnancy, thereby stimulating T cells. We sought to engineer DNA nanocarriers that reproduce this mechanism in human monocytes using clinically translatable materials as a strategy for tumor-associated macrophage (TAM) reprogramming. Methods: A synthetic mimic of fetal cfDNA (scfDNA) was designed, commercially synthesized, and complexed with polyethylenimine (PEI) to form scfDNA-PEI nanoparticles (scfDNA-PEI-NPs) for cytosolic delivery. THP-1 Dual reporter monocytes expressing interferon regulatory factor (IRF)-inducible luciferase were treated with scfDNA-PEI-NPs or human cytotrophoblast derived cfDNA (CTB-DNA) to quantify cGAS-STING-IRF activation. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release to define a non-toxic therapeutic window. Results: scfDNA-PEI-NPs triggered robust cGAS-STING-IRF activation in THP-1 monocytes, exceeding CTB-DNA responses. This response was abolished in cGAS-knockout cells and rescued by a cGAS-independent stimulator of the IRF pathway, confirming cGAS specificity of scfDNA-PEI-NPs. Dose-response analysis showed a > 10-fold separation between cGAS-activating and cytotoxic concentrations, establishing a therapeutic window for myeloid-selective cGAS activation. Conclusions: These findings provide proof-of-concept that pregnancy-inspired DNA nanocarriers can reproduce fetal cfDNA-driven activation of a proinflammatory monocyte phenotype in vitro. This platform lays the groundwork for future work on macrophage-targeted cGAS activation and proinflammatory reprogramming for tumor immunotherapy development. Citation Format: Mathew R. Schnorenberg, Elizabeth Ann L. Enninga, Wendy K. Nevala, Noah A. Stueven, Svetomir N. Markovic, . Pregnancy-inspired DNA nanocarriers reproduce a cGAS-driven pro-inflammatory monocyte phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4307.

Abstract Background: Small cell lung cancer (SCLC) remains an aggressive malignancy with limited treatment options and a 5-year survival rate under 10%. Delta-like ligand 3 (DLL3), expressed in >85% of SCLC but absent from normal tissue, represents an attractive therapeutic target. Existing DLL3-directed antibody-drug conjugates suffer from poor payload stability and limited tumor penetration. To overcome these limitations, we engineered a DLL3-targeted nano-immunoconjugate (NIC) of SN38, the active metabolite of irinotecan, using an albumin-based nanoscaffold. Methods: SN38 was encapsulated in a human serum albumin (HSA) matrix and non-covalently coated with a recombinant anti-DLL3 monoclonal antibody to yield DLL3-SN38-NIC. Stable conjugation was confirmed by HSA-IgG interactions and zeta-potential shift. Binding kinetics were evaluated using Biacore surface plasmon resonance (SPR) and Octet bio-layer interferometry (BLI) employing AHC biosensors to determine DLL3-antibody affinity and specificity. Cytotoxicity was assessed using IncuCyte live-cell imaging across five SCLC cell lines (DMS454, SHP77, NCI-H82, NCI-H889, and NCI-H211) with varying DLL3 expression. Results: DLL3-SN38-NIC retained high-affinity DLL3 binding (KD ≈ 3 × 10-10 M) and demonstrated potent cytotoxicity in SCLC cell lines. NIC formation did not reduce cytotoxic activity compared with free SN38, confirming preserved potency. Cytotoxicity trends were consistent across lines with variable DLL3 expression, and DLL3 antibody alone exhibited negligible cytotoxicity. These findings confirm that nano-immunoconjugate formation maintains drug activity and DLL3 binding; additional in-vivo studies are underway to evaluate pharmacokinetics and tumor selectivity. Conclusions: Albumin-based DLL3-SN38-NICs represent a novel nano-oncologic platform that preserves SN38 potency and achieves high-affinity DLL3 engagement. Ongoing studies will assess in-vivo selectivity, biodistribution, and therapeutic potential in DLL3-positive SCLC and other neuroendocrine malignancies. Citation Format: Mohamed Shanshal, Lin Yang, Liyi Geng, Wendy Nevala, Aaron S. Mansfield, Bharath Wootla, Konstantinos Leventakos, Kyle Korshavn, Svetomir N. Markovic. Albumin-based DLL3-targeted nano-immunoconjugates of SN38 preserve potency and demonstrate high-affinity DLL3 binding in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 449.

The underlying immunopathogenesis of inflammatory arthritis (IA) immune-related adverse event (irAE) remains obscure. Unlike rheumatoid arthritis (RA), where autoantibodies and B cell dysfunction are central features, the contribution of humoral immunity to IA-irAE is unclear. Here, we performed immunophenotyping of peripheral blood from patients with IA-irAE and compared them with patients with seronegative RA, immune checkpoint inhibition–treated patients without irAE, and healthy controls. IA-irAE was marked with increased cytotoxic gene expression and metabolic activation in T cells and reduced CXCR3 and CCR6 expression in CD4 + T cells. Contrary to seronegative RA, patients with IA-irAE displayed no substantial elevation in autoantibody levels or atypical CD11c + CD21 − B cells. IA-irAE was further characterized by elevated levels of interleukin-6 (IL-6), IL-12, and type I interferon, which correlated with the T cell activation phenotypes. Together, our findings define IA-irAE as a disease with certain immunological features distinctive from RA, representing a potentially T cell–driven, autoantibody-independent autoimmunity. These results offer insights into immune tolerance breakdown and therapeutic targeting in irAEs.

Abstract The highest risk for liver allograft rejection is within the first weeks to months after a liver transplant; however, while later-stage mechanisms are well described, the initial window at the moment of alloantigen influx (e.g., ~ 1 h post-reperfusion) remains unmapped. T-cell priming is initiated in lymph nodes, yet these sites are rarely profiled in humans and their relationship to rejection risk is unclear. To address this gap, we profiled paired pre- and ~ 1 h post-reperfusion hepatic draining lymph nodes from liver transplant recipients using multiplexed immunofluorescence and GeoMx whole-transcriptome profiling to identify features that predict rejection versus non-rejection. Early after reperfusion, CD4 + and CD8 + T cells became proximal to macrophages in rejection compared to dendritic cells in non-rejection, which also displayed an immunoregulatory-leaning profile marked by PD-1, IDO1, and CD39, suggesting that macrophages and dendritic cells differentially influence early T-cell alloimmunity. These results provide nodal targets for precise immune modulation.

9503 Background: Neoadjuvant ± adjuvant immunotherapy improves event-free survival relative to adjuvant immunotherapy alone for patients with high-risk resectable stage III melanoma. However, the optimal regimen balancing efficacy and tolerability is not known. T-cell immunoglobulin and ITIM domain (TIGIT) is a promising immune checkpoint but its therapeutic potential in stage III melanoma is underexplored. Methods: In this phase II trial, patients with resectable, macroscopic stage III melanoma received four 21-day neoadjuvant cycles of 1200mg IV atezolizumab (atezo, anti-PD-L1) + 600mg IV tiragolumab (tira, anti-TIGIT), followed by therapeutic lymph node dissection (TLND) and eight 21-day adjuvant cycles of 1200mg IV atezo. Primary endpoints were pathologic response (of all patients initiating neoadjuvant therapy) and recurrence-free survival (RFS) from the time of TLND in patients who were operated on per protocol and received adjuvant therapy. Secondary endpoint was adverse events (AEs); exploratory endpoints included event-free survival (EFS) and distant metastasis-free survival (DMFS). Results: Thirty-four patients, median age 59 years, were accrued and initiated neoadjuvant atezo/tira. 76.5% had >1 metastatic lymph node involved at baseline and 73.5% presented with Stage IIIC disease. All 34 patients were evaluable for AEs, pathologic response and EFS. Four patients were diagnosed with metastatic disease during neoadjuvant treatment, while 30 had TLND per protocol and 28 received adjuvant treatment and were evaluable for RFS and DMFS. Major pathologic responses (MPR, ≤10% viable tumor) were observed in 16/34 patients (47.1%, Table). With 19.9 months median follow-up from registration, 12-month EFS was 72.0% (95% CI 57.9 to 89.5%). With 16 months median follow-up from operation, 12-month RFS was 73.3% (n=28, 95% CI 56.9 to 94.5%), while 12-month DMFS was 86.0% (n=28, 95% CI 72.2 to 100%). In the 16 patients with an MPR, 2 did not receive adjuvant treatment and were followed for 33.3 and 10.8 months without recurrence/death. In the remaining 14 patients, 12-month RFS and 12-month DMFS were both 91.7% (95% CI 77.3 to 100%). 5 patients (14.7%) experienced any grade 3+ AE with 2 (5.9%) at least possibly related to the neoadjuvant regimen. Conclusions: Among patients with high-risk resectable stage III melanoma, neoadjuvant atezo/tira was a promising regimen with a favorable safety profile and warrants further study. Identification of predictive biomarkers will allow for optimization of neoadjuvant therapy for individual patients. Clinical trial information: NCT03554083 . Pathologic response. N=34 Major Pathologic Response 16 (47.1%) Pathologic complete response (no viable tumor) 13 (38.2%) Near-pathologic complete response (0.1−10% viable tumor) 3 (8.8%) Pathologic partial response (>10.0−50% viable tumor) 2 (5.9%) Pathologic non-response (>50% viable tumor) 12 (35.3%) No per protocol operation 4 (11.8%)

Abstract Background: The tumor microenvironment (TME) plays a pivotal role in ovarian cancer (OC) progression and prognosis, while also contributing to biological differences between primary and metastatic tumors. This study examines OC TMEs to uncover associations with clinical prognosis and tumor biology by analyzing paired primary and metastatic tumors from the same patients. Methods: This study focused on 57 stage-IIIC/IV OC patients treated at the Mayo Clinic, all of whom underwent surgical debulking and adjuvant therapies. All patients provided informed consent, and the study received IRB approval. A tissue microarray (TMA) was specifically designed and constructed, containing paired primary and metastatic tissue cores from the same patients. 147 tissue cores from the constructed TMA were profiled using a 35-antibody panel, with 17 antibodies for cell phenotyping and 18 for functional state characterization. Profiling was performed using Cell DIVE, a multiplexed imaging solution at the single-cell level developed by Leica Microsystems. After cell segmentations and classifications, detailed spatial niche and regional analyses were performed at the single-cell level. Immune cell populations, as well as their TME distributions, were characterized to conduct prognostic associations and contrast primary and metastasis differences. Results: A total of 1,292,988 total cells (after excluding low-density signals and ambiguous classifications) were analyzed, with a median of 8,033 cells per core. Using spatial neighborhood analysis, 12 distinct spatial niches were identified with specific cell types and/or unique functional states. Among these, a macrophage- and fibroblast-enriched niche at the tumor-stroma interface was associated with improved overall survival (p=0.008). Another rare Treg-enriched niche, along with other immune cells, demonstrated protective effects, particularly in patients with residual macroscopic tumors after debulking surgery (p=0.05), while no significant effects were observed in those without visible residual disease (p=0.8). When comparing primary and metastatic tumors, metastatic lesions exhibited significantly increased tumor mass (p=2e-4) and a notable reduction in helper T cells (p=0.04) in tumor areas, as well as significantly increased fibroblast cells in tumor-stroma interfacing regions (p=0.002), reflecting distinct immunosuppressive and tumor-promoting shifts. Conclusion: This study reveals spatial and cellular differences in primary and metastatic OC TMEs, distribution differences linked to prognosis and surgical outcomes. These findings emphasize the potential of spatially resolved TME profiling to refine adjuvant and neoadjuvant therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6266.

Background Neoadjuvant treatment has become standard for patients with high-risk operable stage III melanoma, but the optimal regimen is unknown. Targeted therapy approaches yield high pathological response rates, while immunotherapy regimens show favorable recurrence-free survival (RFS). NeoACTIVATE was designed to address whether a neoadjuvant combination of both targeted therapy and immunotherapy might leverage the benefits of each. Methods We tested neoadjuvant treatment with 12 weeks of vemurafenib, cobimetinib, and atezolizumab for patients with BRAF-mutated (BRAFm) melanoma (cohort A) and cobimetinib and atezolizumab for patients with BRAF-wild-type (BRAFwt) melanoma (cohort B), regimens which we have shown generate a substantial major pathological response. After therapeutic lymph node dissection, patients received 24 weeks of adjuvant atezolizumab. Here, we report survival outcomes and their association with biomarkers assayed among the gut microbiome and peripheral blood immune subsets. Results With 49 months median follow-up, the median RFS was not reached for cohort A and was 40.8 months for cohort B. At 24 months after operation, 2 of 14 cohort A patients and 4 of 13 cohort B patients had experienced distant relapse. Key findings from correlative analyses included diversity, taxonomic and functional metagenomic gut microbiome signals associated with distant metastasis-free survival at 2 years. Notably, we observed a strong correlation between low microbial arginine biosynthesis (required for T-cell activation and effector function) and early distant recurrence (p=0.0005), which correlated with taxonomic differential abundance findings. Peripheral blood immune monitoring revealed increased double-positive (CD4+CD8+) T cells in patients with early recurrence. Conclusions Neoadjuvant treatment with cobimetinib and atezolizumab±vemurafenib was associated with a low rate of distant metastasis in patients with high-risk stage III melanoma. Freedom from early distant metastasis was highly associated with taxonomic differences in gut microbiome structure and with functional pathway alterations known to modulate T cell immunity. Identification of predictive biomarkers will permit optimization of neoadjuvant therapy regimens for individual patients. Trial registration number NCT03554083.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Common treatments following surgical resection include PD-1-targeting checkpoint inhibitors (pembrolizumab), as 20% of tumors are PD-L1 positive with or without systemic chemotherapy. Over the last several years, our laboratory has developed nano-immune conjugates (NIC) in which hydrophobic chemotherapy drugs like paclitaxel (PTX) and SN38, the active metabolite of irinotecan, are made water soluble by formulating them into albumin-based nanoparticles (nab) that are hydrophobically linked to various IgG1 monoclonal antibodies, creating an antigen-targetable nano-immune conjugate. To date, we have successfully tested PTX containing NICs linked to either VEGF- or CD20-targeted antibodies in two phase I clinical trials against multiple relapsed ovarian/uterine cancer or non-Hodgkin’s lymphoma, respectively. Herein, we describe a novel NIC created with either PTX or SN38 that is coated with anti-PD-L1-targeting antibodies for the treatment of a preclinical model of TNBC. In vitro testing suggests that the chemotherapy drug and antibody retain their toxicity and ligand binding capability in the context of the NIC. Furthermore, both the PTX and SN-38 NIC demonstrate superior anti-tumor efficacy relative to antibody and chemotherapy drugs alone in a PD-L1 + MDA-MB-231 human TNBC xenograft model, which could translate clinically to patients with TNBC.