Abstract Although the role of neutrophils in modulating antitumor T-cell responses has been extensively studied, their direct effects on tumor cells remain less well understood. In this study, we investigated whether neutrophils have the capacity to directly kill tumor cells independently of T cells. We found that anti-CD40–based therapy, when combined with IL10 receptor blockade, initiates a Batf3-dependent pathway in which IL12 and IFNγ secretion results in oncolytic neutrophil activity. Using a combination of microscopy, single-cell, and functional assays, we observed that killing of tumor cells by neutrophils is dependent on physical contact and degranulation. This degranulation-mediated killing is associated with an atypical dynamic invasive neutrophil phenotype. In line with our preclinical findings, our phase I trial of anti-CD40 shows that circulating IL12, IFNγ, and IL10 increase in response to anti-CD40, whereas our phase Ib/2 PRINCE study shows that lower circulating IL10 is associated with favorable overall survival (OS) specifically among anti-CD40–treated patients. Finally, we found that neutrophil expansion with granulocyte colony-stimulating factor is associated with improved OS, specifically in patients treated with anti-CD40, suggesting that this pathway may be amenable to therapeutic intervention in patients with advanced cancer.

Alterations in tumor infiltrating lymphocytes and myeloid cell populations following treatment with PSMAi-C’ dots and ICB in the Hi-Myc model (Day 10 post treatment). (a) Schematic of prostate cancer development and treatment before immunophenotyping of Hi-Myc GEM models. (b – o) Bar charts illustrating distinct T cell (b – i) and myeloid (j – o) populations harvested from Hi-Myc tumor-bearing mice (n = 3/group) treated with a multi-dose regimen (three doses every three days) of saline, ICB, PSMAi-C’dots or Dual Tx. Tumors were harvested 10 days after the final dose and dissociated to single cells, which were analyzed via multi-color flow cytometry. Data in (b – o) are presented as mean ± s.e.m. and a 1-way ANOVA with Tukey's multiple comparisons test was performed. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns, not significant.

Non-targeted C’ dots induce tumor-specific inflammatory responses in the Hi-Myc prostate cancer model. Ex vivo gene expression profiling of (a) tumor and (b) spleen harvested from Hi-Myc mice 4 days after the final dose of vehicle or non-targeted particles. Specific classes of genes (i.e., iron/ferroptosis-related, DAMPs, antigen presentation, and immune-related) were profiled. Transcripts were normalized to Gapdh and represented as fold changes, with significant changes indicated by values above 2 (dashed lines). Gene expression changes represented by bar plots at (c) 24 h and (d) 96 h post-particle administration in Hi-Myc mice, shown as log2 fold change transcript alterations relative to saline; significance indicated by values > 1 (solid lines).

PSMAi-C' dots trigger proinflammatory responses in prostate cancer and immune cells. (a) Gene expression levels, as fold changes over controls, using qRT-PCR and (b) normalized cytokine and chemokine expression levels by Proteome Profiler in Myc-CaP cells and supernatants, respectively, following incubation with 15 uM of PSMAi-C’ dots over 72 h. (c) IFN-a/b reporter B16 cell luminescence (OD620) after incubation with supernatants from particle-treated and untreated Myc-CaP cells. Percentage (%) of surface-expressed (d) IFNGR1, (e) MHC-I, (f) PD-L1, and (g) CD73 on vehicle- and particle-treated Myc-CaP cells by flow cytometry at 72-hour post-exposure. (h) Flow cytometry-based analyses of the %M1 and %M2 markers in vehicle- and particle-treated bone marrow-derived macrophages (BMDMs) over 72 h. (i) IFN-a/b reporter B16 cell luminescence (OD620) after incubation of BMDMs with supernatants from (h). Percentage of CD8+ T cell-specific (j) IFN-g and (k) TNF-a secretion from splenocytes by flow cytometry following a 72-hour incubation with or without PSMAi-C’ dots. (L) T cell cytotoxicity (expressed as luciferase (luc+) activity) following co-culture of mouse T cells with luc + Myc-CaP cells, with and without PSMAi-C’ dots for 48 h. All samples were run in triplicate. Numerical data are presented as mean ± s.e.m. 1-way ANOVA with Kruskal-Wallis test was performed in (a). Unpaired t-tests were performed in (d–g) and (i–l). 2-way ANOVA with Šídák's multiple comparisons test was performed in (c) and (h). *p < 0.05, **p < 0.01, ****p < 0.001.

Mouse kidney parvovirus (MKPV) causes inclusion body nephropathy, resulting in clinical signs and mortality in immunodeficient mice and subclinical infection in immunocompetent mice. While late-stage renal lesions and viral replication have been characterized, a comprehensive multisystemic investigation of MKPV infection from the initial to the late stages of infection has not been conducted. Our goal was to investigate lesions and viral replication in all major organs at multiple stages of MKPV infection in immunocompetent C57BL/6NCrl (B6) and Crl: CD1(ICR) (CD1) mice and immunodeficient NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Following experimental oronasal inoculation with MKPV, mice were evaluated at 15 time points from 1.5 to 112 days post-inoculation (DPI) by histology and in situ hybridization for MKPV RNA on all major organs, as well as immunohistochemistry for markers of immune cells and renal tubular injury. In all strains, the gastrointestinal mucosa was the initial site of viral replication beginning at 3 DPI and persisting through the study without associated lesions. In B6 and CD1 mice, viral replication was first detected in renal tubules on 28 and 14 DPI, respectively, and lymphoplasmacytic tubulointerstitial nephritis was first evident on 63 and 49 DPI, respectively. B6 mice displayed the lowest levels of renal viral replication and lesion severity. In contrast, renal viral replication was highest in NSG mice; the virus was first detected on 42 DPI and in association with tubular degeneration from 63 DPI. Electron microscopy on kidney tissues of infected mice revealed parvoviral virions, nuclear replication, and assembly compartments for the first time.

Serum cytokine/chemokine analyses demonstrate PSMAi-C’ dots maintain a safe inflammatory profile relative to controls in the presence and absence of ICB. Proteome profiles of detectable cytokines and chemokines in serum from mice treated with saline, PSMAi-C' dots, ICB, and Dual Tx (a) 4 days and (b) 10 days following the final dose. All data are presented as mean ± s.e.m. (c) Corresponding body weights of mice over time (n = 5 per cohort). 1-way ANOVA with Tukey's multiple comparisons test was performed (***p < 0.005).

SIRPα is a well-characterized inhibitory receptor on myeloid immune cells. However, human and mouse melanoma cells can also express high levels of SIRPα. Whether and how melanoma cell-intrinsic SIRPα contributes to tumor progression and anti-tumor immunity remains underexplored. Here, we identify a role of tumor cell-intrinsic SIRPα in suppressing immune recruitment and activation. SIRPα deletion in melanoma cells enhances tumor control and increases immune infiltration. Transcriptomic analyses reveal that loss of tumor cell-intrinsic SIRPα upregulates the chemokine CXCL10 in both human and mouse melanoma cells. Notably, Cxcl10 knockdown in SIRPα-deficient melanoma partially rescues tumor growth and reduces CD8+ T cell infiltration, mirroring the phenotype of SIRPα-expressing tumors and indicating that tumor cell-intrinsic SIRPα promotes immune evasion by suppressing Cxcl10-mediated T cell recruitment. Our study uncovers an unrecognized mechanism of SIRPα-mediated immune suppression and highlights SIRPα silencing as a potential therapeutic strategy to enhance immune infiltration and T cell-mediated tumor control across SIRPα-expressing cancers.

Background Large animal models of bladder cancer are lacking. Objective This study aimed to develop and characterize a transgenic porcine model of bladder cancer (BC) using Oncopigs expressing Cre-inducible KRAS G12D and TP53 R167H mutations. Methods Eleven female Oncopigs underwent tumor induction via three cystoscopic inoculation procedures: Procedure I (N = 3, 1 inoculation/pig), chemical dissolution of the glycosaminoglycan layer with N-Dodecyl-β-d-Maltoside DDM followed by adenoviral Cre-recombinase (AdCre) instillation; Procedure II (N = 4, 3 inoculation/pig), mechanical mucosal denudation followed by AdCre instillation; and Procedure III (N = 4, 3 inoculation/pig), cystoscopy-guided submucosal injection of AdCre. Animals were clinically monitored throughout follow-up (14–28 days). Tumor development was assessed on cystoscopy and ultrasonography, and pathologically, immunohistochemically (IHC), and genomically characterized. Results All pigs remained clinically healthy. Tumors developed at 59% (16/27) of inoculation sites: nine (33%) were neoplastic and seven (26%) were inflammatory. Procedure I achieved 100% neoplastic tumors and produced both non-muscle invasive (71%) and muscle-invasive (29%) tumors. Procedure II achieved 50% neoplastic tumors, all of which were muscle invasive (100%). Procedure III generated only inflammatory tumors. Histologically, neoplastic tumors were pathologically interpreted as urothelial cell carcinomas with sarcomatoid differentiation, with IHC confirming the presence of both epithelioid and sarcomatoid features with abundant mixed leukocytic infiltrates. Genomic analyses verified Cre-induced alterations alongside other mutations seen in human BC. Conclusions We herein demonstrate an efficient and reproducible method for developing autochthonous neoplastic bladder tumors in Oncopigs that resemble human bladder cancer of varying stages. This large animal model facilitates the evaluation of novel surgical and intravesical therapies in BC.

Complete gene list for 6 cell types (Related to Figure 5F)

Although the role of neutrophils in modulating antitumor T-cell responses has been extensively studied, their direct effects on tumor cells remain less well understood. In this study, we investigated whether neutrophils have the capacity to directly kill tumor cells independently of T cells. We found that anti-CD40-based therapy, when combined with IL10 receptor blockade, initiates a Batf3-dependent pathway in which IL12 and IFNγ secretion results in oncolytic neutrophil activity. Using a combination of microscopy, single-cell, and functional assays, we observed that killing of tumor cells by neutrophils is dependent on physical contact and degranulation. This degranulation-mediated killing is associated with an atypical dynamic invasive neutrophil phenotype. In line with our preclinical findings, our phase I trial of anti-CD40 shows that circulating IL12, IFNγ, and IL10 increase in response to anti-CD40, whereas our phase Ib/2 PRINCE study shows that lower circulating IL10 is associated with favorable overall survival (OS) specifically among anti-CD40-treated patients. Finally, we found that neutrophil expansion with granulocyte colony-stimulating factor is associated with improved OS, specifically in patients treated with anti-CD40, suggesting that this pathway may be amenable to therapeutic intervention in patients with advanced cancer.

Volumetric visualization of tumor cell and interacting neutrophil shown in Figure 7A. Tumor cell is shown in green, interacting neutrophil in purple, and overlapping volume in red.

Pharmacokinetic and biodistribution studies in Myc-CaP tumor-bearing mice following i.v.-injection of 89Zr-DFO-PSMAi-C’ dots. (a) PD-10 elution profile of 89Zr-DFO-PSMAi-C’ dots. Pure radiolabeled product elutes between 2.5-5 mL. (b) Stability of 89Zr-DFO-PSMAi-C’ dots in human (n = 3 samples) and mouse serum (n = 3 samples) relative to phosphate buffered saline (PBS, n = 3 samples). (c) In vivo targeted PET imaging of 89Zr-DFO-PSMAi-C’ dots in one of four representative Myc-CaP tumor-bearing mouse over a 72-hour period post-injection. (d) Tumor- and organ-specific time-activity curves and activity-to-background ratios were obtained from all mice (n = 4) in the study (c). (e) Biodistribution (represented as injected dose per gram tissue, %ID/g) of 89Zr-DFO-PSMAi-C’ dots in mice from (c) 72 hr post-injection of particle tracer. Data in (b) and (d – f) are presented as mean ± s.e.m.

Background Sacroiliac joint (SIJ) dysfunction accounts for the etiology of pain in 15%-30% of low back pain cases. Some patients with conservative treatment-refractory SIJ dysfunction undergo radiofrequency (RF) ablation of the SIJ for prolonged pain relief. This procedure involves placing up to 12 RF probes in what is an invasive, resource-intensive, and time-consuming process. High-intensity focused ultrasound is an alternative neuroablative technique that is non-invasive and potentially less cumbersome. MRI-guided high-intensity focused ultrasound (MRgHIFU) had previously been successfully applied to SIJ ablation in a swine model, and more recently had been trialed in humans. However, ultrasound-guided high-intensity focused ultrasound (USgHIFU) of the SIJ may be a more practical and rapid alternative to MRIgHIFU.Methods This was a prospective technology efficacy and safety study in a swine model. Three Yorkshire pigs underwent bilateral SIJ ablation using a proprietary USgHIFU prototype. Post procedure, treatment efficacy was assessed using clinical evaluation of pain and changes in ambulation, gross inspection of lumbosacral necropsy and pathology sections, and histology.Results Post anesthetic monitoring for 72 hours showed no signs of gait abnormalities or perceived pain in the swine models. Of the primary sacral spine targets, histological specimen review suggested successful lesioning of 37/54 sites (68.5%), specifically in the targeted areas that were visualized under ultrasound. Of the successful lesion zones, 22/37 (59.5%) included nerve lesions, 34/37 (91.9%) included muscle lesions, 34/37 (91.9%) included periosteum lesions, and 20/21 (95.2%) included bony lesions.Conclusions The preliminary study thus demonstrates that USgHIFU can create targeted contiguous strip lesions along the SIJ and lead to thermal necrosis of the posterior sacral network without causing additional neurological damage or damage to adjacent muscle tissue or bone outside of target areas.

Volumetric visualization of interaction shown in Supplemental Video 1

Volumetric visualization of interacting neutrophil (inner box) and tumor cell shown in Supplemental Video 2.

Contact depicted in circle C of Figure 7A between tumor cell and interacting neutrophil.