Malignant glioma is considered invariably recalcitrant, thus necessitating development of new therapeutic approaches. Spontaneously arising canine glioma exhibits important molecular and pathological similarities to malignant pediatric glioma, making it an invaluable model for testing novel therapies. We evaluated safety and activity of RNA lipid particle aggregates (LPAs) designed to provoke cancer immunogenicity in 10 dogs with histologically confirmed glioma. Vaccine manufacturing was feasible in all subjects and generally well tolerated. Median survival time was 123 and 155 days in dogs receiving three (Group A) or four (Group B) vaccines, respectively; this was increased from historical reporting of subjects receiving palliative care. Responding animals had a robust cytokine response following infusions. In animals receiving non-specific RNA-LPAs, there was localization of messenger RNA (mRNA) payloads to the brain tumor microenvironment and a rapid shift in immunologic gene signatures consistent with a viral response. These data demonstrate the ability to induce rapid viremic response in the tumor microenvironment following a single peripherally administered mRNA vaccine. Since RNA-LPAs are amenable to repeat boosts, these formulations may overcome challenges of tumor immunosuppression and therapeutic access and are currently under evaluation in first-in-human trials (NCT04573140, NCT06389591).

BackgroundUltraviolet radiation (UVR) affects local cutaneous and systemic immunity acutely. The wavelength, pattern and intensity of UVR exposure, individual skin phototype and immune state of individuals modulate the impact of UVR systemically. Local cutaneous immunity after UVR leads to immunosuppression that impacts melanoma. However, the effects of systemic UVR-induced changes on solid cancer therapy are not known.MethodsWe investigated the impact of repeated UVR exposure on systemic immunity and immune checkpoint blockade (ICB) tumor responses in colorectal cancer and melanoma mouse models.ResultsAnimals exposed to chronic UVR exhibit decreased ICB response, which is mediated by systemic factors. Repeated UVR exposure expanded systemic lymphocyte populations and contracted total systemic myeloid cells. Specifically, UVR expanded peripheral blood CD4+ regulatory T cells (Tregs), which in turn led to greater Treg infiltration and immunosuppression in colorectal and skin cancer, and colorectal ICB-resistant tumors expressed unique pathways of ICB resistance due to systemic UVR. The response to ICB was restored with systemic pharmacological depletion of Tregs. In preliminary human data, there is an association between the molecular evidence of repeated UVR exposure in dermal fibroblasts and higher systemic Tregs.DiscussionOur data indicate that patients with melanoma and other cancers on immunotherapy should avoid repeated sun exposure.

PurposeA recently published phase 2 neoadjuvant trial in patients with early-stage non-small cell lung cancer (NSCLC) (NCT02818920) evaluated the potential efficacy of pembrolizumab administration in the absence of chemotherapy. This communication reports on conventional and distribution-based immune profiling efforts in efforts to identify novel biomarkers predictive of benefit.MethodsPatients with stage 1B-3A NSCLC received two cycles of pembrolizumab (P), followed by surgical resection of the remaining tumors (NCT02818920). Banked peripheral blood mononuclear cells (PBMCs) were analyzed at baseline and following the second dose of P. Resected tumors were disaggregated, and cells were viably cryopreserved. Based on pathologic examination of the tumors, patients were categorized as major pathologic responders (MPR; ≤10% viable tumor present), or non-MPR (>10% viable tumor present). High-parameter immune phenotyping by flow cytometry was performed on all available tumor and PBMC specimens, and results were expressed using both conventional phenotypic frequency analyses as well as a novel distribution-based biomarker identification strategy aimed at discovery of patterns associated with MPR.ResultsConventional, frequency-based flow cytometric immune phenotyping of participant tumor microenvironments and PBMC revealed several MPR-associated trends, only a few of which reached statistical significance. The distribution-based biomarker identification strategy greatly enhanced the discovery of statistically significant cell types and patterns of change associated with MPR.ConclusionsThis novel, distribution-based analytic framework identified MPR-associated immune cell subsets in baseline PBMC that were not evident using conventional frequency-based immune profiling. Profiling the microenvironment of MPR-associated tumors revealed statistically significant distributional differences among highly expressed cellular markers on CD8+ cells.

BackgroundDespite comparable survival benefit has been obtained, the drug resistance remarkably reduced lenvatinib clinical efficacy. Here, we aimed to identify the potential mechanism by which cysteine and glycine-rich protein 2 (CSRP2) regulates the development of hepatocellular carcinoma (HCC) and participates in the resistance to lenvatinib.MethodsWe harnessed RNA sequencing, multiplex immunofluorescence staining, and hydrodynamic tail vein (HTV) injection HCC model to systematically explore the function of CSRP2 in HCC progression. To precisely delineate how immunosuppressive macrophages, influenced by CSRP2-regulated C-C motif chemokine ligand 28 (CCL28) signaling, respond to lenvatinib-induced cytotoxicity, we established an in vitro co-culture system and conducted functional cytotoxicity assays.ResultsUsing RNA sequencing, multiplex immunofluorescence staining and HTV injection HCC mouse model, we identified CSRP2 as one of the most significantly upregulated genes in HCC tissues. CSRP2 overexpression drives anti-lenvatinib resistance by inducing high levels of tumor-associated macrophages (TAMs) infiltration and reshaping an immunosuppressive microenvironment. Then flow cytometry, mass spectrometry and chromatin immunoprecipitation were conducted to clarify the underlying mechanism of CSRP2. We showed CSRP2 promotes phosphorylation of activating transcription factor 2 (ATF2) at Thr69/71, leading to the transcriptional activation of CCL28 expression. HCC-derived CCL28 recruits TAMs to drive immunosuppression and anti-lenvatinib tolerance. BI6901, a potent and selective CCR10 antagonist, blocked TAMs recruitment and enhanced T-cell activation. Combining CCR10 inhibition improved the therapeutic benefit of anti-lenvatinib in HCC.ConclusionsThese results illustrate that CSRP2 regulates the tumor microenvironment to promote HCC growth and drive lenvatinib tolerance via the CSRP2/ATF2/CCL28 axis. Targeting this pathway could synergize with lenvatinib to treat HCC more effectively.

Immune checkpoint inhibition (ICI) has become a first-line treatment strategy for advanced hepatocellular carcinoma (HCC). However, treatment efficacy remains varied, and there are no reliable biomarkers of response. We used phage immunoprecipitation sequencing technology to measure circulating viral and bacterial antibodies as a biomarker of ICI response. The XGBoost Cox-proportional hazard model identified 23 viral and bacterial strains associated with survival. An antigen score developed from the 23 reactive microbial peptides significantly predicted overall survival (HR: 81.7, [95% CI]: 9.88 to 10,690.96, p<0.0001) and a high antigen score was predictive of progressive disease (HR: 4.03, [95% CI]: 0.78 to 20.87, p=0.09). The time-dependent area under the curve was 0.8 at 1 year. Findings were validated in an independent cohort and confirmed the antigen score association with survival (HR: 2.38, [95% CI 0.98 to 5.76, p=0.055). Our results suggest a unique microbial reactivity profile may serve as a potential biomarker of ICI response in patients with HCC.

BackgroundPatients with colon cancer liver metastases (CCLM) frequently exhibit poor responses to immunotherapy, a phenomenon attributed in part to an immune desert tumor microenvironment. This study aimed to comprehensively characterize the immune landscape in primary colon cancers and their matched liver metastases via single-cell transcriptome analysis, with the goal of identifying potential immunotherapeutic targets.MethodsTumor specimens from patients with CCLM were subjected to single-cell RNA sequencing. Immune subpopulations were profiled with emphasis on exhausted T cells (Tex)—including both CD8+ and CD4+ ANK3+ subsets—as well as on a distinct stress response T-cell subset (TSTR) defined by high HSPA1A/HSPA1B expression. In parallel, we performed assessments of the phenotype and prognostic impact of SPP1high myeloid cells, along with in vitro assays to examine their role in modulating T-cell number and function.ResultsLiver metastatic lesions exhibited a significantly elevated infiltration of Tex compared with primary tumors. Notably, Tex cells exhibited upregulated expression of exhaustion-related marker genes such as ANK3, ZBTB20, ETV6, and CAMK4, which were markedly downregulated in TSTR cells. TSTR was identified as an intermediate developmental state between effector and exhausted T cells in patients with CCLM, suggesting that TSTR cells represent a distinct state from exhausted T cells. Furthermore, myeloid cells expressing high levels of secreted phosphoprotein 1 (SPP1), along with apolipoprotein C-I and apolipoprotein E, were associated with poor prognosis in patients with CCLM. In vitro studies revealed that Macro_SPP1high cells diminished T-cell populations and triggered a stress response state in both CD4+ and CD8+ T cells via the SPP1/CD44/PI3K/AKT signaling pathway in a CD44-dependent manner. Importantly, combination treatment with anti-SPP1 and anti-programmed cell death protein-1 antibodies significantly inhibited liver metastasis growth, enhanced dendritic cell maturation, decreased M2-polarized macrophages, and restored T-cell infiltration and function.ConclusionsThese findings reveal a previously unrecognized relationship between Macro_SPP1high cells and HSPA1Ahigh/HSPA1Bhigh T cells in driving CCLM progression, suggesting a potential synergistic therapeutic approach that could boost immune checkpoint treatment efficacy in patients with CCLM.

The inconsistent immunotherapy response among lesions in patients with multiple primary lung cancer (MPLC) remains poorly understood, presenting a significant challenge for effective treatment. In this study, we conducted a comprehensive multiomics analysis of all lesions from a patient with MPLC who exhibited varied responses to neoadjuvant chemoimmunotherapy. Further verification was conducted through external single-cell data and multiplex immunohistochemistry of three cases of MPLC.Notably, tertiary lymphoid structures (TLSs) were observed across all nodules, regardless of response, suggesting possible TLS impairment in non-responsive nodules. Cell neighborhood (CN) analysis revealed that type II alveolar epithelial cell (AT2) cell–positive CNs were prevalent in non-responsive nodules, while AT2-negative CNs appeared in responsive nodules, strongly associating AT2 cell presence with a reduced therapeutic response. Spatial colocalization analysis further showed that AT2 cells surrounding TLSs upregulated immunosuppressive markers on B cells within TLSs. The mechanism of this suppressive effect was further unveiled that macrophage migration inhibitory factor (MIF), secreted by AT2 cells, binds to sialic acid acetylesterase (SIAE) receptors on B cells by single-cell RNA sequencing analysis, which were validated in four additional non-responsive nodules from three other patients with MPLC. Multiomics analysis revealed AT2 cells exert immunosuppressive effects by inhibiting B cells within TLS through MIF-SIAE signaling axis in patients with MPLC. These findings offered new perspectives for tailored immunotherapy for patients with MPLC.

High densities of tertiary lymphoid structures (TLSs) are associated with improved clinical outcomes in various malignancies, including human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). However, the role of TLSs in shaping antitumor immunity in HPV-induced cervical cancer (CESC) remains unclear. Therefore, we analyzed the density, composition, and prognostic impact of TLSs in patients with CESC as well as patients with HNSCC. Multiplex immunofluorescence, immunohistochemistry, and spatial transcriptomics were used to analyze TLS density and composition in HNSCC and CESC tissue sections with respect to patient prognosis. The spatial approach was supplemented by flow cytometry-based analysis of the polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) phenotype in freshly resected primary tumor tissues. Although both indications were associated with HPV infection, we confirmed a positive correlation between TLS density and improved overall survival only in patients with HNSCC. The TLS composition differed markedly between HNSCC and CESC samples, with a shift toward high regulatory T cell (Treg) and PMN-MDSC abundance in CESC samples. The highest Treg and PMN-MDSC levels were observed in patients with CESC who died of the disease. CESC-infiltrating PMN-MDSCs showed high arginase 1 expression, which correlated with diminished T-cell receptor (TCR)ζ chain expression in CESC-infiltrating T cells. Additionally, the high number of PMN-MDSCs in TLSs was associated with the absence of HPV-specific T cells in CESC. Unlike in HNSCC, the composition of TLSs, rather than their quantity, was associated with the overall survival of patients with CESC. High numbers of Tregs and PMN-MDSCs infiltrating immature TLSs prevail in patients with CESC who succumbed to the disease and seem to affect tumor-specific immune responses.

Circulating tumor DNA (ctDNA) is a promising intermediate end point for oncology drug development, potentially accelerating regulatory approvals by providing early insights into treatment response. However, challenges remain in standardizing ctDNA assessment, including optimal blood collection timing and defining molecular response (MR) cutoffs. The ctDNA for Monitoring Treatment Response (ctMoniTR) project, led by Friends of Cancer Research, aggregates patient-level data from clinical trials to evaluate associations between ctDNA changes and overall survival (OS). This analysis included four randomized clinical trials of patients with advanced non-small cell lung cancer (aNSCLC) treated with either anti-programmed death (ligand) 1 (anti-PD(L)1) therapy (with or without chemotherapy) or chemotherapy alone. MR was assessed using three predefined per cent-change thresholds in ctDNA levels (≥50% decrease, ≥90% decrease, and 100% clearance). ctDNA samples were analyzed at two timepoints: an early window (T1, up to 7 weeks post-treatment initiation) and a later window (T2, 7-13 weeks post-treatment initiation). Multivariable Cox proportional hazards models and time-dependent analyses were used to evaluate associations between ctDNA changes and OS. A total of 918 patients were included. In the anti-PD(L)1 group, ctDNA reductions at both T1 and T2 were significantly associated with improved OS across all MR thresholds. In the chemotherapy group, associations were weaker at T1 but became more pronounced at T2. Patients with MR at both T1 and T2 had the strongest OS associations. Overall, the results suggest that T2 had marginally stronger association with OS than T1. This study supports the potential of ctDNA as an intermediate end point in aNSCLC, with MR at both early (T1) and later (T2) timepoints showing significant associations with OS. Differences in ctDNA dynamics between treatment modalities highlight the importance of considering the timing of blood collection. Further research is needed to determine the optimal time window for assessing ctDNA response. Prospective trials and trial-level meta-analyses will be critical to validating ctDNA as a regulatory-grade intermediate end point for oncology drug development.

BackgroundImproving the efficacy of anti-programmed death 1 (PD-1) monoclonal antibody (mAb) therapy remains a major challenge for cancer immunotherapy in non-small cell lung cancer (NSCLC). Gut microbial metabolites can influence immunotherapy efficacy.MethodsELISA was used to compare the serum 5-hydroxyindoleacetic acid (5-HIAA) level in patients with NSCLC. Humanized mice were constructed to observe the effect of 5-HIAA on immunotherapy. RNA-seq and flow cytometry were used to analyze the effect of 5-HIAA on tumor-infiltrating lymphocytes. The effects of phenelzine (Phe) and Akkermansia muciniphila (AKK) on 5-HIAA synthesis, antitumor immunity and immunotherapy efficacy were analyzed. Finally, the synergistic effect of Phe combined with AKK on anti-PD-1 mAb was observed.ResultsHere we found that 5-HIAA, which is regulated by gut microbiota, has increased concentrations in the serum of non-responders to immunotherapy. Supplementation of 5-HIAA inhibited the efficacy of anti-PD-1 mAb and tumor infiltration of CD8+ T cells. The use of monoamine oxidase inhibitor (MAO-I) Phe inhibited the synthesis of 5-HIAA, then improved the efficacy of anti-PD-1 mAb. In addition, supplementation of AKK can also decrease 5-HIAA in serum. Finally, the combination of Phe and AKK maximally inhibited 5-HIAA synthesis and improved immunotherapy efficacy.ConclusionsOur investigations reveal that alterations in gut microbial composition leading to increased 5-HIAA synthesis can negatively impact CD8+ T cell functionality and the success of immunotherapy. The combination of Phe and AKK supplementation holds potential for optimizing immunotherapy efficacy.