Schimke, Jill M.

CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.

Abstract Purpose: AB160 is a 160-nm nano-immunoconjugate consisting of nab-paclitaxel (ABX) nanoparticles noncovalently coated with bevacizumab (BEV) for targeted delivery into tissues expressing high levels of VEGF. Preclinical data showed that AB160 resulted in greater tumor targeting and tumor inhibition compared with sequential treatment with ABX then BEV. Given individual drug activity, we investigated the safety and toxicity of AB160 in patients with gynecologic cancers. Patients and Methods: A 3+3 phase I trial was conducted with three potential dose levels in patients with previously treated endometrial, cervical, and platinum-resistant ovarian cancer to ascertain the recommended phase II dose (RP2D). AB160 was administered intravenously on days 1, 8, and 15 of a 28-day cycle (ABX 75–175 mg/m2, BEV 30–70 mg/m2). Pharmacokinetic analyses were performed. Results: No dose-limiting toxicities (DLT) were seen among the three dose levels tested. Grade 3/4 toxicities included neutropenia, thromboembolic events, and leukopenia. DL2 (ABX 150 mg/m2, BEV 60 mg/m2) was chosen as the RP2D. Seven of the 19 patients with measurable disease (36.8%) had confirmed partial responses (95% confidence interval, 16.3%–61.6%). Pharmacokinetic analyses demonstrated that AB160 allowed 50% higher paclitaxel dosing and that paclitaxel clearance mirrored that of therapeutic antibodies. Conclusions: The safety profile and clinical activity of AB160 supports further clinical testing in patients with gynecologic cancers; the RP2D is DL2 (ABX 150 mg/m2, BEV 60 mg/m2).

Abstract Background: Neuropilin 2 (NRP2) is a co-receptor that enhances signaling of growth factor and cytokine receptors and is expressed in tumor and microenvironment (TME) cells in melanoma and other tumors. Neuropilin 2 expression in tumor cells is associated with more aggressive tumors. Expression in tumor associated macrophages enhances efferocytosis and immunologically silent tumor cell death. Methods: Whole excisional lymph node biopsies were obtained from 10 treatment naïve patients with metastatic cutaneous melanoma who subsequently received anti-PD1 treatment. A single 5mm formalin-fixed paraffin embedded tissue section was used to assess a panel of 39 analytes by cyclic multiplex immunofluorescence (MxIF). Fields of view (~1mm2, n=288) were selected from pathologist-annotated regions of the tumor core, tumor-immune interface and adjacent lymphoid tissue. Pixel-based single cell segmentation and a supervised classifier approach was applied to resolve 12 distinct tumor, stromal and immune cell phenotypes and functional states (NRP2 positive or negative) in 1.5 million cells. Results: Tumor recurrence was observed in 6 patients (relapsed), whereas 4 patients were recurrence-free (non-relapsed) during follow up. Among the different cell types, NRP2 expression was more frequent in macrophages (44%), tumor cells (40%), dendritic cells (40%), followed by Tregs (31%) and T helper cells (29%), and less frequent in neutrophils (24%), cytotoxic T cells (23%) and B cells (15%). In colocalization permutation analysis, NRP2 expressing macrophages clustered away from tumor cells in non-relapsed patients while a random spatial distribution of NRP2 expressing macrophages with respect to tumor cells was observed in relapsed patients. In comparing the odds of tumor cell interactions with NRP2-expressing macrophages to those with NRP2 non-expressing macrophages, we found in the tumor:stromal interface (10% tumor cells) that cells from both non-relapsed and relapsed patients were more likely to interact with NRP2 non-expressing macrophages (OR = 0.73, 95% CI = [0.71, 0.75] and OR = 0.56, 95% CI = [0.55, 0.58] for non-relapsed and relapsed patients, respectively). However, in the tumor core (90% tumor cells), tumor cells from relapsed patients had significantly higher odds of interacting with NRP2 expressing macrophages, (OR = 1.15, 95% CI = [1.12, 1.18]), and this was not seen for non-relapsed patients (OR = 0.68, 95% CI = [0.66, 0.71]). In FOVs dominated by tumor cells (75% tumor), tumor cells from relapsed patients had 4.97 times the odds of expressing NRP2 compared to tumor cells from non-relapsed patients (95% CI = [2.23, 11.09], p = 0.0001). Conclusions: These spatial analytics suggest complex interactions among NRP2 expression in tumor and TME of melanoma determining risk for relapse with anti-PD1 treatment. Citation Format: Anastasios Dimou, Raymond M. Moore, Caitlin Ward, Alexey Leontovich, Ruifeng Guo, Chen Wang, Shankar Suman, Betty Dicke, Jill M. Schimke, Noah A. Stueven, Chathu A. Atherton, Wendy Nevala, Svetomir N. Markovic. Spatial analysis of neuropilin 2 expression in the microenvironment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2905.

Introduction: Nanotechnology for cancer therapy aims to increase drug delivery to tumor sites while lowering toxicity. AR160 is a novel 160 nm nano-immunoconjugate consisting of commercial grade nab-paclitaxel (NP) nanoparticles non-covalently coated with rituximab (R) for targeted delivery into tissues expressing CD20. The drug delivery system and clinical grade product was developed in the laboratory of Svetomir N. Markovic, M.D.,Ph.D. Single agent rituximab at a dose of 375 mg/m 2 in relapsed patients with aggressive lymphomas is not utilized and has limited activity in patients with multiple relapses. Taxanes (Younes 1995, Wilson 1995) have modest clinical activity as a single agent with overall response rates of 23% and 17%, respectively, when administered at doses of 200 mg/m 2 and 140 mg/m 2 in previously treated patients with non-Hodgkin lymphoma (NHL). We hypothesized that utilizing the targeted ability of rituximab that the AR160 taxane nanoparticle would have activity in relapsed B-cell NHL and offer a new class of agents for relapsed B-cell NHL. Methods: A 3+3 phase I clinical trial was conducted in patients with relapsed refractory non-Hodgkin lymphoma to determine the maximum tolerated dose of AR160 administered intravenously on days 1, 8, and 15 of a 28-day cycle. Patients were required to have had prior systemic treatment with no other therapy with clinical benefit. Patients with aggressive lymphoma were required to have a stem cell transplant if transplant eligible. AR160 was prepared on-site in the Mayo Clinic Cancer Center Research Pharmacy, and the product was reconstituted by mixing the two agents delivering as one combined infusion at a specific concentration. The starting dose level (DL1) was NP at 100mg/m 2 with R at 40 mg/m 2. These doses were significantly lower than standard doses. Disease evaluations were conducted after every 2 treatment cycles using CT based criteria from the revised 2014 Lugano Classification or PET scans. Patients were treated until disease progression or intolerability. Samples were collected for pharmacokinetic (PK) studies. Results: Nine (7 males and 2 females) patients with a median age of 61 (range 55-84 years) were enrolled (2 follicular lymphoma (FL), 1 previously transformed from FL to diffuse large B-cell lymphoma (DLBCL) followed by FL relapse, 1 high-grade B-cell lymphoma with MYC and BCL2 rearrangements transformed from FL, 1 transformed FL to DLBCL, 2 high-grade B-cell lymphoma with MYC and BCL2 rearrangements, 1 high-grade B-cell lymphoma NOS and 1 mantle cell lymphoma. The median number of prior treatments was 4 (range 3-16). At dose level 1, all 3 of the first 3 patients experienced grade 3 or 4 neutropenia (C1D15, C4D1, and C1D15) with the C4D1 a grade 4 and unlikely related. After the first three patients, de-escalation to dose level -1 was implemented per protocol with NP at 75 mg/m 2 and rituximab at 30 mg/m 2. There was one patient with a grade 3 non-hematological adverse event all possibly related to treatment (anorexia, diarrhea, and enterocolitis). There were 6 partial responses (71%), 1 complete response (14%) with high-grade B-cell lymphoma with MYC and BCL2 rearrangement, 1 stable disease with mantle cell lymphoma and 1 patient had progressive disease. Six of the 9 patients were alive with subsequent therapies. PK evaluation is pending. Conclusion: AR160 therapy is a novel drug delivery system incorporating significantly lower doses of paclitaxel and rituximab that is safe and demonstrates promising clinical activity in patients with low-grade and high-grade previously treated B-cell lymphomas. This principle of treatment is applicable to other disorders. Further clinical testing is warranted in these patient populations.

Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion, including the downregulation of HLA-I, potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet, the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune, and vasculature components across 323 fields of view. Single-cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core fields of view of responders. Integrating single-cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the tumor-centric cellular neighborhood (TCCN) based on anatomic subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytic framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy. Significance: Findings from this work propose a novel approach to resolving clinical heterogeneity of the TIME by objectively quantifying the cellular interactions occurring in metastatic melanoma lymph node tissue utilizing multiplex immunofluorescence. This study provides an analytic and biologically derived unit of measure, the TCCN which is customizable for studying critical paracrine interactions within spatially preserved tissue of various cancers and across the spectrum of multiplex imaging modalities.

Triiodothyronine (T3) has a major impact on the regulation of energetics and mitochondrial (mt) function. We determined whether chronic T3 administration alters the age‐related decline in mt function. Ten young (Y, 8 mo) and ten old (O, 27 mo) F344/BN rats were randomized to receive T3 (200 mg/d) or placebo for 14d via subcutaneous osmotic pump. Because OT3 rats decreased food consumption by 39%, we added ten pair fed rats (OPF). T3 treatment caused a significant weight loss (Y, −11.97%; O, −17.49%), while energy expenditure increased in both Y (50.86%) and O (54.40%), but decreased in PF (17.55%). Fat and muscle loss were similar in Y and O in response to T3 (fat, −52.53%(Y) vs −51.69% (O); muscle, −21.50% (Y) vs −21.03% (O) in soleus and −16.70% (Y) vs −17.35% (O) in plantaris), whereas these changes were minor with OPF (fat, −10.4%; soleus −1.69% ; plantaris 7.69%). While mt ATP production and citrate synthase activity in heart and soleus muscle were lower in O, these measures increased equally in Y and O in response to T3. In the soleus, mt DNA abundance increased in Y (54.39%) and O (23.36%) and UCP3 expression increased 10‐fold in both age groups. Gene transcripts levels of PGC1α, COX3&4 and NADH4, UCP2& 3 increased in soleus of T3 rats. In conclusion, T3 similarly increased energy expenditure, weight loss and mt biogenesis in both Y and O rats, despite differences in food intake. NIH grant PO1 AG09531 and Mayo Foundation

Aging is associated with a concomitant decline in non exercise activity (NEA) and mitochondrial ATP production rate (MAPR) in skeletal muscle. To determine whether caloric restriction (CR) could prevent the decline in MAPR and NEA we studied young (Y, 7 month‐old, n=8), old (O, 27 month‐old, n=7) and old caloric‐restricted F344/BN rats (OCR, 27 month‐old, 40% CR from 16 weeks of age, n=7). Energy expenditure (kcal/d/gBW) was lower in O (0.311±0.005) than Y (0.365±0.004) and was higher in OCR (0.341±0.008) (P<0.05). Muscle mass (mg/gBW) of O was lower than Y, and Y and OCR are similar (Y=0.96±0.18, O=0.71±0.16, OCR=1.03±0.04; P<0.05 OCR vs O). Citrate synthase activity and MAPR were lower in both O and OCR than Y (P<0.05). UCP3 protein level was higher in soleus and plantaris muscles from O rats than Y and OCR (p<0.005). CR lowered UCP3 expression in skeletal muscles to the level in Y rats (p<0.005 OCR vs O). CR also lowered expression of mRNA levels of NADH4 (p<0.005 OCR vs O). NEA levels were lower in O and OCR rats than in Y (p<0.05 Y vs O and OCR). In conclusion, the decline in mitochondrial functions during aging can be counteracted by CR but an age‐related decline in activity levels could not be prevented. Funded by NIH RO1 AG09531 and Mayo Foundation.

e22020 Background: The combination of nab-paclitaxel (NP) and bevacizumab (BEV) in patients with MM has shown promising clinical activity. AB160 is a 160 nm nano-immunoconjugate of NP nanoparticles non-covalently coated with BEV for targeted delivery into high VEGF expressing tissues. Preclinical data showed that AB160 improved tumor targeting/ tumor inhibition more than NP followed by BEV. Methods: A 3+3 phase I trial was conducted in patients (pts) with MM who had prior systemic treatment for metastatic disease to determine the maximum tolerated dose of AB160 administered intravenously on days 1, 8 and 15 of a 28-day cycle. Dose level 1 (DL1) was 125 mg/m2 NP /50 mg/m2 BEV. Dose limiting toxicities (DLT) included grade (G) 4 neutropenia or anemia, PLT < 25,000, serum creatinine ≥ 2 times baseline, G2-4 neurologic toxicity or G3-4 non-hematologic toxicities. Tumor evaluations (RECIST) were conducted every 8 weeks. Treatment continued until progression or intolerability. Results: 21 pts (11 ♀) aged 36-78 years old were enrolled. One of the first 3 pts on DL1 developed a G2 colonic perforation; this was considered a DLT. One of the next 3 pts on DL1 had a DLT: G4 neutropenia. Of the 3 pts on DL-1 (100 mg/m2 NP/40 mg/m2 BEV), 2 had no DLTs and 1 died of sepsis after C1D1 dose. Enrollment was suspended until an amendment modifying the eligibility criteria was approved by the IRB. The trial reopened. One of the 4 pts on DL-1 and 1 of the 5 pts on DL1 had a DLT: G3 pain and G3 fatigue, respectively. Enrollment ended after 2 of the 3 pts on DL2 (150 mg/m2 NP/ 60 mg/m2 BEV) developed G4 neutropenia. Thus, MTD is DL1. A median of 3 cycles was administered. Treatment ended due to progression (9), intolerability (9), refusal (2) and death (1). There were no objective tumor responses. Common G3-4 toxicities were: neutropenia (33%) and thromboembolic events (19%). Conclusions: AB160 was found to have insufficient clinical benefit in patients with previously treated MM to justify further development. However, parallel phase I testing in gynecologic cancers suggests clinical benefit (abstract #300225). Clinical trial information: NCT02020707.