Immunosuppressive Tumor Microenvironment Research Articles

Overcoming Resistance to Immune Checkpoint Blockade in Liver Cancer with Combination Therapy: Stronger Together?

Primary liver cancer, represented mainly by hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA), is one of the most common and deadliest tumors worldwide. While surgical resection or liver transplantation are the best option in early disease stages, these tumors often present in advanced stages and systemic treatment is required to improve survival time. The emergence of immune checkpoint inhibitor (ICI) therapy has had a positive impact especially on the treatment of advanced cancers, thereby establishing immunotherapy as part of first-line treatment in HCC and CCA. Nevertheless, low response rates reflect on the usually cold or immunosuppressed tumor microenvironment of primary liver cancer. In this review, we aim to summarize mechanisms of resistance leading to tumor immune escape with a special focus on the composition of tumor microenvironment in both HCC and CCA, also reflecting on recent important developments in ICI combination therapy. Furthermore, we discuss how combination of ICIs with established primary liver cancer treatments (e.g. multikinase inhibitors and chemotherapy) as well as more complex combinations with state-of-the-art therapeutic concepts may reshape the tumor microenvironment, leading to higher response rates and long-lasting antitumor immunity for primary liver cancer patients.

Dual-Targeted Self-Adjuvant Heterocyclic Lipidoid@Polyester Hybrid Nanovaccines for Boosting Cancer Immunotherapy.

Tumor vaccines have demonstrated a modest response rate, primarily attributed to their inefficient delivery to dendritic cells (DCs), low cross-presentation, DC-intrinsic immunosuppressive signals, and an immunosuppressive tumor microenvironment (TME). Here, draining lymph node (DLN)-targeted and tumor-targeted nanovaccines were proposed to address these limitations, and heterocyclic lipidoid (A18) and polyester (BR647) were synthesized to achieve dual-targeted cancer immunotherapy. Meanwhile, oligo hyaluronic acid (HA) and DMG-PEG2000-Mannose were incorporated to prepare dual-targeted nanovaccines encapsulated with STAT3 siRNA and model antigens. The nanovaccines were designed to target the DLN and the tumor, facilitating the delivery of cargo into the cytoplasm. These dual-targeted nanovaccines improved antigen presentation and DC maturation, activated the stimulator of interferon genes (STING) pathway, enhanced the pro-apoptotic effect, and stimulated antitumor immune responses. Additionally, these dual-targeted nanovaccines overcame immunosuppressive TME, reduced immunosuppressive cells, and promoted the polarization of tumor-associated neutrophils from N2 to N1. Among the four dual-targeted nanovaccines that induced robust antitumor responses, the heterocyclic lipidoid@polyester hybrid nanovaccines (MALO@HBNS) demonstrated the most promising results. Furthermore, a combination strategy involving MALO@HBNS and an anti-PD-L1 antibody exhibited an immensely powerful anticancer role. This work introduced a dual-targeted nanovaccine platform for antitumor treatment, suggesting its potential combination with an immune checkpoint blockade as a comprehensive anticancer strategy.

Necroptosis-Mediated Synergistic Photodynamic and Glutamine-Metabolic Therapy Enabled by a Biomimetic Targeting Nanosystem for Cholangiocarcinoma.

Targeted delivery of glutamine metabolism inhibitors holds promise for cholangiocarcinoma therapy, yet effective delivery vehicles remain a challenge. This study reports the development of a biomimetic nanosystem, termed R-CM@MSN@BC, integrating mesoporous organosilicon nanoparticles with reactive oxygen species-responsive diselenide bonds for controlled release of the glutamine metabolism inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and the photosensitizer Ce6. Erythrocyte membrane coating, engineered with Arg-Gly-Asp (RGD) peptides, not only enhanced biocompatibility but also improved tumor targeting and tissue penetration. Upon laser irradiation, R-CM@MSN@BC executed both photodynamic and glutamine-metabolic therapies, inducing necroptosis in tumor cells and triggering significant immunogenic cell death. Time-of-flight mass cytometry analysis revealed that R-CM@MSN@BC can remodel the immunosuppressive tumor microenvironment by polarizing M1-type macrophages, reducing infiltration of M2-type and CX3CR1+ macrophages, and decreasing T cell exhaustion, thereby increasing the effectiveness of anti-programmed cell death ligand 1 immunotherapy. This strategy proposed in this study presents a viable and promising approach for the treatment of cholangiocarcinoma.

IL-3-driven T cell-basophil crosstalk enhances antitumor immunity.

Cytotoxic T lymphocytes (CTLs) are pivotal in combating cancer, yet their efficacy is often hindered by the immunosuppressive tumor microenvironment, resulting in exhaustion. This study investigates the role of interleukin (IL)-3 in orchestrating anti-tumor immunity through CTL modulation. Intratumoral CTLs undergo a progressive decline in IL-3 production, which is correlated with impaired cytotoxic function. Augmenting IL-3 supplementation, through intraperitoneal administration of recombinant IL-3, IL-3-expressing tumor cells, or IL-3-engineered CD8+ T cells, confers protection against tumor progression, concomitant with increased CTL activity. CTLs are critical in this therapeutic efficacy as IL-3 demonstrates no impact on tumor growth in Rag1 knockout mice or following CD8+ T cell-depletion. Rather than acting directly, CTL-derived IL-3 exerts its influence on basophils, synergistically amplifying anti-tumor immunity within CTLs. Introducing IL-3-activated basophils retards tumor progression, whereas basophil depletion diminishes the effectiveness of IL-3 supplementation. Furthermore, IL-3 prompts basophils to produce IL-4, which subsequently elevates CTL IFN-γ production and viability. Notably, the importance of basophil-derived IL-4 is evident from the absence of benefits of IL-3-supplementatation in IL-4 knockout tumor-bearing mice. Overall, this research unveils IL-3-mediated CTL-basophil crosstalk in regulating anti-tumor immunity, and offers the prospect of harnessing IL-3 sustenance as a promising approach for optimizing and enhancing cancer immunotherapy.

Why does circadian timing of administration matter for immune checkpoint inhibitors' efficacy?

Tolerability and antitumour efficacy of chemotherapy and radiation therapy can vary largely according to their time of administration along the 24-h time scale, due to the moderation of their molecular and cellular mechanisms by circadian rhythms. Recent clinical data have highlighted a striking role of dosing time for cancer immunotherapy, thus calling for a critical evaluation. Here, we review the clinical data and we analyse the mechanisms through which circadian rhythms can influence outcomes on ICI therapies. We examine how circadian rhythm disorders can affect tumour immune microenvironment, as a main mechanism linking the circadian clock to the 24-h cycles in ICIs antitumour efficacy. Real-life data from 18 retrospective studies have revealed that early time-of-day (ToD) infusion of immune checkpoint inhibitors (ICIs) could enhance progression-free and/or overall survival up to fourfold compared to late ToD dosing. The studies involved a total of 3250 patients with metastatic melanoma, lung, kidney, bladder, oesophageal, stomach or liver cancer from 9 countries. Such large and consistent differences in ToD effects on outcomes could only result from a previously ignored robust chronobiological mechanism. The circadian timing system coordinates cellular, tissue and whole-body physiology along the 24-h timescale. Circadian rhythms are generated at the cellular level by a molecular clock system that involves 15 specific clock genes. The disruption of circadian rhythms can trigger or accelerate carcinogenesis, and contribute to cancer treatment failure, possibly through tumour immune evasion resulting from immunosuppressive tumour microenvironment. Such emerging understanding of circadian rhythms regulation of antitumour immunity now calls for randomised clinical trials of ICIs timing to establish recommendations for personalised chrono-immunotherapies with current and forthcoming drugs.

DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer

ObjectiveHighly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments...

Updated results from first-in-human phase 1 dose-escalation trial of TAK-102, a GPC3-targeted armored CAR T cells, in patients with advanced solid tumors.

2543 Background: Glypican 3 (GPC3) is a member of the glypican family of heparan sulfate proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol anchor. High GPC3 expression rates are reported in numerous cancer types with a high unmet medical need, including hepatocellular carcinoma (HCC). TAK-102 is a GPC3-targeted autologous chimeric antigen receptor T cell (CAR-T) immunotherapy armored with IL-7 and CCL19. The addition of IL-7 and CCL19 to the construct was designed to support the expansion of memory subsets and persistence of CAR-T cells. We hypothesized TAK-102 would help overcome the challenges associated with an immunosuppressive tumor microenvironment that limit the activity of nonarmored CAR-T therapies in solid tumors. Methods: The first-in-human, Phase 1 dose escalation study evaluated TAK-102 in patients (pts) with GPC3-expressing solid tumors who were refractory or intolerant to standard treatments. TAK-102 was administered via a single infusion to 3 dose cohorts after lymphodepleting chemotherapy (LDC; consisting of fludarabine and cyclophosphamide): dose level (DL) 1 (starting cohort), 1x107 CAR+ cells/body; DL2, 1x108 CAR+ cells/body; DL3, 5x108 CAR+ cells/body. Objectives included evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, cellular kinetic (CK) parameters, tumor markers, cytokine/chemokine and antitumor activity based on RECIST 1.1. Results: Eleven pts were enrolled and infused TAK-102 (DL1: 3 pts, DL2: 3 pts, DL3: 5 pts): 1 gastric neuroendocrine carcinoma, 2 liposarcoma, 8 HCC. Five pts achieved stable disease (SD), HCC (GPC3 H-Score: 36) . In patients achieving SD, the greatest reduction in tumor size was 26.4%. No DLTs or neurotoxicity were observed. Six pts experienced cytokine release syndrome (Grade1: 5 pts, Grade2: 1 pt); all cases were manageable. AFP was measured as a tumor marker for HCC. Among 8 pts with HCC, 4 had SD after treatment with TAK-102 and 3 showed a decrease or stabilization of AFP levels corresponding to their clinical status. CK profiles for pts were assessed by flow cytometry and qPCR-based assays. Overall, there was improvement in TAK-102 exposure (Cmax, AUC) when escalating from DL1 to DL2. There was a slight decrease in Tmax from DL2 to DL3. Homeostatic cytokine (IL-7) spiked post-LDC and showed no further increase after TAK-102 infusion across all the DLs. There was dose-dependency observed in peak CCL19, IFN-γ and IL-6 levels. Conclusions: TAK-102, an armored CAR-T, is well tolerated and has a manageable safety profile with some early signs of anti-tumor activity. For CK, TAK-102 exposure (Cmax, AUC) showed improvement from DL1 to DL2, and slight decrease in Tmax from DL2 to DL3. Dose-dependency was observed in peak CCL19, IFN-γ and IL-6 levels, which may point towards increased signal of activity from DL1 to DL3. Clinical trial information: NCT04405778 .

Phase I clinical study of FC084CSA, a high selective AXL kinase inhibitor that ameliorates tumor microenvironment and augments the effectivity of immunotherapy.

e15116 Background: NSCLC patients with STK11 gene mutations could not benefit from immunotherapy, according to the data of multiple clinical trials. This is associated with immunosuppressive tumor microenvironment (TME) due to elevated AXL expression, and deficiency in pro-inflammatory cytokines and effector T cells. AXL inhibitor bemcentinib (formerly BGB324) has been found to improve the effectiveness of immunotherapy of immune checkpoint inhibitors. FC084CSA is a high selective AXL inhibitor. Methods: This is an ongoing first-in-human, phase I study of FC084CSA in patients with advanced solid tumors. The primary objective is to evaluate the safety and tolerability of FC084CSA. The secondary objectives include preliminary efficacy and pharmacokinetics. Results: As of January 10th, 2024, 9 patients with a median age of 60 years (44-73) were enrolled in 4 different dose levels: 100mg QD, 200mg QD, 400mg QD and 600mg QD. All patients had ≥ 2 prior lines of prior therapy. No dosing-limiting toxicities were observed. 8 (88.9%) patients experienced treatment-related adverse events (TRAEs). Most TRAEs were grade 1 or 2 and only one grade 3 TRAE was observed. The most common TRAEs (≥20%) included increased blood lactate dehydrogenase (44.4%), elevated gamma-glutamyl transferase (33.3%), proteinuria (33.3%), anemia (33.3%), increased aspartate aminotransferase (22.2%), weight loss (22.2%), increased blood creatinine (22.2%) and vomiting (22.2%). A total of 8 patients had at least 1 post-baseline tumor assessment. Objective response rate (ORR) was 0% and disease control rate (DCR) was 62.5% (5/8). Conclusions: FC084CSA was well tolerated in patients with previously heavily treated solid tumors. Clinical trial information: NCT06231550 .

Antagonizing vasoactive intestinal peptide (VIP) receptors with Muc16CD-directed armored CAR T cells for pancreatic cancer.

2558 Background: Chimeric antigen receptor (CAR) T therapies for pancreatic ductal adenocarcinoma (PDAC) still face significant immunosuppressive obstacles in the tumor microenvironment (TME). We hypothesize that an optimal CAR T cell for PDAC combines targeting an ideal tumor-associated antigen (TAA) and overcomes unique immunosuppression in the PDAC TME. The retained ectodomain of Muc16 (Muc16CD) is a known TAA in ovarian cancer but has yet to be explored in pancreatic cancer. Vasoactive intestinal peptide (VIP) is an emerging checkpoint pathway for T cell function, which expresses VIP receptors (VIPR) and is abundantly expressed by PDAC. In this work, we present a novel armored CAR T cell that targets Muc16CD and antagonizes VIPRs (CAR/VIPRa) to overcome the immunosuppressive PDAC TME. Methods: Patient expression data was assessed based on data generated by the TCGA Research Network. Primary human T cells from healthy donors or PDAC patients were retrovirally transduced to express Muc16CD-directed CARs with or without secretion of novel, potent VIPR antagonist peptides. In vivo, PDAC PDX tumors were engrafted into SCID/Beige mice and treated with CAR T cells. Results: PDAC tumors have significantly increased expression of Muc16 compared to normal pancreas tissue and patients with high Muc16 expression have a significantly decreased overall survival. PDAC patient-derived tumors show robust expression of both Muc16CD and VIP. CAR/VIPRa T cells reveal that VIPR antagonism metabolically reprograms CAR T cells and drives a memory-rich product. CAR/VIPRa T cells are less activated and less exhausted by the manufacturing process, which lends to better viability and a metabolically quiescent phenotype at baseline. These distinct features allow CAR/VIPRa T cells, when antigen-stimulated, to have enhanced activation and expansion with repeated stimulation. To investigate clinical relevance, CAR/VIPRa T cells manufactured from PDAC patient blood are also significantly enriched for memory phenotypes. In vivo, CAR/VIPRa T cells have enhanced expansion, phenotype, infiltration, and persistence, which ultimately reduces PDAC tumor burden. In a patient-derived PDAC preclinical mouse model where CAR T is typically ineffective, CAR/VIPRa T cells significantly reduce tumor burden. Conclusions: This work demonstrates Muc16CD as a clinically relevant TAA target for CAR T therapy in PDAC. Furthermore, antagonizing the previously undescribed VIP checkpoint pathway in CAR T cells produces enhanced phenotypic and functional profiles. Collectively, this data demonstrates that novel CAR/VIPRa T cells create an advantageous cellular therapy product capable of treating PDAC. The long-term goal of this work is translating CAR/VIPRa T cells for the treatment of PDAC and expanding these preclinical findings of cellular therapies for other VIP-abundant tumors.

Immunomodulatory alpha neutrophils: The first-in-class neutrophil progenitor-based allogeneic immuno-cell therapy with cytotoxic and immunomodulatory functionality for the treatment of solid tumours.

e14523 Background: Adoptive cell therapy for the treatment of cancer has largely focused on the administration of autologous engineered αβ T cells. Although successful in some haematological malignancies, the solid tumour microenvironment (TME) poses significant challenges to the efficacy of CAR-T cells, including physical barriers, heterogeneous antigen expression or antigen escape and immunosuppressive mediators. Remodeling the immunosuppressive solid TME is pivotal to the success of immunotherapies. Recent studies have highlighted key roles for neutrophils in tumour control and in facilitating successful responses to cancer immunotherapy, with neutrophil functional heterogeneity within the TME becoming increasingly appreciated. Methods: At LIfT Biosciences we have developed the first-in-class, stem cell-derived immuno-cell therapy with anti-cancer neutrophil properties called Immunomodulatory Alpha Neutrophils (IMANs). IMANs are derived from CD34+ stem cells expanded and differentiated in proprietary media compositions in a simple and scalable GMP-compliant manufacturing process. IMANs are designed to recapitulate those neutrophil states that are both cytotoxic and capable of immunomodulating the solid TME for long-lasting tumour control. Off-the-shelf, allogeneic, innate IMANs have the potential to provide major clinical benefits in the treatment of multiple solid tumours. Results: IMANs multimodal mechanisms of action (MoA) have been demonstrated in different state-of-the-art solid tumour models, including co-culture with patient-derived xenograft organoids (PDX-O) and with 3D ex vivo patient tissues (tumour-on-a-chip). Direct antigen-independent cytotoxicity towards PDX-O from a range of solid tumour indications has been demonstrated. Additionally, and fundamental to IMANs MoA is their ability to migrate to the solid TME and recruit and activate recipients’ anti-tumour immune responses. We have demonstrated IMANs ability to rapidly migrate into the solid TME and promote recruitment of patients’ immune cells leading to enhanced tumour cell death using tumour-on-a-chip technology accompanied by daily live cell imaging. Mechanistically, co-culture with IMANs enhanced expression of co-stimulatory receptors on T and NK cells, such as OX40 and 4-1BB, as well as promoting secretion of TME-modulating factors, such as CXCL10. Furthermore, co-culture with IMANs enhanced secretion of IFN-γ by activated tumour-infiltrating lymphocytes. Conclusions: These cytotoxic and immunomodulatory functions enable IMANs to remodel the TME, leaving the solid tumour more susceptible to host immune responses.

The BTLA-HVEM axis restricts CAR T cell efficacy in cancer.

The efficacy of T cell-based immunotherapies is limited by immunosuppressive pressures in the tumor microenvironment. Here we show a predominant role for the interaction between BTLA on effector T cells and HVEM (TNFRSF14) on immunosuppressive tumor microenvironment cells, namely regulatory T cells. High BTLA expression in chimeric antigen receptor (CAR) T cells correlated with poor clinical response to treatment. Therefore, we deleted BTLA in CAR T cells and show improved tumor control and persistence in models of lymphoma and solid malignancies. Mechanistically, BTLA inhibits CAR T cells via recruitment of tyrosine phosphatases SHP-1 and SHP-2, upon trans engagement with HVEM. BTLA knockout thus promotes CAR signaling and subsequently enhances effector function. Overall, these data indicate that the BTLA-HVEM axis is a crucial immune checkpoint in CAR T cell immunotherapy and warrants the use of strategies to overcome this barrier.

Neuroinflammation in Glioblastoma: The Role of the Microenvironment in Tumour Progression.

Glioblastoma (GBM) stands as the most aggressive and lethal among the main types of primary brain tumors. It exhibits malignant growth, infiltrating the brain tissue, and displaying resistance toward treatment. GBM is a complex disease characterized by high degrees of heterogeneity. During tumour growth, microglia and astrocytes, among other cells, infiltrate the tumour microenvironment and contribute extensively to gliomagenesis. Tumour-associated macrophages (TAMs), either of peripheral origin or representing brain-intrinsic microglia, are the most numerous nonneoplastic populations in the tumour microenvironment in GBM. The complex heterogeneous nature of GBM cells is facilitated by the local inflammatory tumour microenvironment, which mostly induces tumour aggressiveness and drug resistance. The immunosuppressive tumour microenvironment of GBM provides multiple pathways for tumour immune evasion, contributing to tumour progression. Additionally, TAMs and astrocytes can contribute to tumour progression through the release of cytokines and activation of signalling pathways. In this review, we summarize the role of the microenvironment in GBM progression, focusing on neuroinflammation. These recent advancements in research of the microenvironment hold the potential to offer a promising approach to the treatment of GBM in the coming times.

Enrichment of glioblastoma tumor microenvironment in a GZMK+ T cell population.

2065 Background: Glioblastoma (GBM) bears a survival estimate below 10% at 5 years, despite standard chemoradiation treatment. Trials evaluating immune checkpoint blockade (ICB) in gliomas have been disappointing so far, dealing with the unique immunosuppressive tumor microenvironment (TME) of the brain. Nevertheless, we have clinical evidence of ICB efficacy for brain metastases (BrM) of immunosensitive tumors. Even if both GBMs and BrM share the same organ-specific microenvironment, it is clear that disease-specific immune characteristics instruct differential response to ICB. Methods: By using advanced deep learning algorithms to combine public and in-house-generated single-cell RNA sequencing profiles, we analyzed immune infiltrate characteristics of 602949 CD45+ cells from 76 GBM, 30 BrM from different types of extracranial tumors (13 NSCLC, 12 melanoma, 2 ovarian, 1 breast, 1 colorectal and 1 renal carcinoma) and 3 normal brain samples. The analysis was restricted to newly diagnosed GBM, excluding recurrent and pre-treated cases. CD8+ cells underwent segregation into clusters based on log2 normalized expression levels of CD3E and CD8A (with expression values greater than 0). The data integration utilized DESC, a deep learning model founded on autoencoders, and subsequent examination was conducted within the Scanpy framework. Cluster analysis was executed employing the Leiden algorithm. Differentially expressed genes (DEGs) were pinpointed across clusters using the Wilcoxon test, with False Discovery Rate (FDR) correction carried out through the Benjamini-Hochberg procedure. Results: From CD45+CD3+ cells, we isolated CD8+ T cells and identified 7 informative CD8+ T cells clusters. Based on DEGs, we could appreciate T cells clusters that were also recapitulated in other extracranial malignancies. The most abundant subset was composed of GZMK+ effector T cells (Cluster 0: GZMK, GZMM, CD44, KLRG1, IL7R, GZMA, CXCR3), followed by putative tumor-specific tissue-resident memory cells (Cluster 1: GZMB, LAG3, CTLA4, TIGIT, CXCL13, ITGAE, ENTPD1, PDCD1, CD27, HLA-DRB1), stem-cell memory-like T cells (Cluster 3: IL7R, CCR7, JUNB, KLF2, TCF7) and a less represented cluster characterized by high expression of genes belonging to the HSP family (Cluster 4: HSPA1, HSPH1, HSPE1, HSPD1, IFN). Interestingly, while the majority of the identified clusters was equally represented in the 3 different tissues, the GZMK+ effector T cells cluster (Cluster 0) was found to be more represented in GBM compared to BrM and normal brain tissue. Conclusions: A GZMK+ effector T cells subpopulation, specifically enriched in GBM, has been poorly characterized in its function and spatial localization. Our data highlight the importance of studying the composition of the immune infiltrate in GBM with multi-omics approaches in order to uncover deep mechanisms of resistance to ICB and eventually provide hints on potential therapeutic targets.

Ferroptosis-induced immunomodulation with biometabolic MOF@COF nanovaccine for self-boosting anti-tumor immunotherapy

Ferroptosis, a regulated cell death pathway, presents a compelling approach for augmenting anti-tumor immunity, addressing challenges associated with immune escape and inadequate immunogenicity linked to apoptosis resistance. In this study, we introduce a biometabolic MOF@COF nanovaccine (NMCAH) designed to evoke self-amplifying anti-tumor responses through ferroptosis-mediated immune stimulation. The nanovaccine demonstrates the capacity to induce the generation of reactive oxygen species and deplete glutathione within tumors, initiating immunogenic ferroptosis. Simultaneously, it facilitates the polarization of macrophages toward the M1 phenotype and the maturation of CD8+ T cells. Upon priming, these immune cells autonomously amplify the response in a self-reinforcing manner. Notably, IFN-γ secretion by CD8+ T cells reinforces macrophage polarization and induces secondary ferroptosis via ACSL4 up-regulation. This intricately coordinated interplay between ferroptosis and adaptive immunity holds significant promise in overcoming the immunosuppressive tumor microenvironment. Our study establishes a versatile nanotherapeutic platform for advancing cancer immunotherapy by leveraging the immunogenic characteristics of regulated cell death.

Genomic and tumor microenvironment dynamics of brain metastases in breast cancer.

1018 Background: Brain metastases (BM) in breast cancer (BC) have a poor prognosis, with median survival at 7.2-13 months. Interplay between genomic evolution of cancer cells and development of BM is obscure. We sought to identify genomic aberrations associated with BM. Methods: We included 14095 BC samples: 429 BM, 7858 extra-cerebral metastases (ECM) and 5808 primary tumors (PT) were analyzed by next generation sequencing of DNA (NextSeq, 592 genes and NovaSeq, WES) and RNA (NovaSeq, WTS) (Caris Life Sciences, AZ). Tumor mutational burden-high (TMB-H) was defined as ≥10 mt/MB. Cut-off for PD-L1 immunohistochemistry positivity was ≥2 intensity and >5% staining (SP142). Global loss of heterozygosity (gLOH) used a cut-off of ≥ 16%. PAM50 was evaluable for a subset (n=9724, 69%) with WTS data. Statistical significance determined using chi-square and Mann-Whitney U test and adjusted for multiple comparisons (q<0.05). Results: Median age was lower in BM than ECM and PT (55 vs 62 vs 59 years, q<0.05). BM subtype was triple negative (TNBC, 40.1%), HR+/HER2- (30.5%), or HER2+ (29.4%) vs. ECM primarily HR+/HER2- (65.4%) and PT HER2+ (52.5%). BM were Basal-like (35.3%) or HER2-enriched (35.3%), whereas ECM were Luminal B (47.2%) and PT were Basal-like (34.4%) or Luminal B (34.6%). The Table summarizes molecular findings. In HR+/HER2- BC, BM were associated with TMB-H vs. ECM and PT (q<0.05). BM had higher GATA3-mt and ESR1-mt compared to PT (q<0.05). In HER2+ BC, BM had lower PD-L1+ (11.8% vs 39.3%) and higher ESR1-mt than PT. BM had higher ERBB2-amp (93.5% vs 75.2%) and RARA-amp (27.3% vs 6.7%) than PT (q<0.05). TNBC BM had higher proportion of TMB-H tumors but lower PD-L1+ (20.8% vs 50.7%) than PT. BM had increased gLOH than ECM (59.2% vs 36.2%). PIK3CA was lower in BM vs. ECM. In all subtypes, BM, when compared to ECM and PT respectively, were associated with decreased AR+ (HR+/HER2-: 57.7% vs 80.8% vs 85.7%; HER2+: 54.4% vs 77.1% vs 81%; TNBC: 15.1% vs 27.7% vs 23.1%), increased dendritic cell infiltration (HR+/HER2-: 3.2% vs 2.6% vs 2.5%; HER2+: 3.85% vs 2.41% vs 2.36%; TNBC: 3.96% vs 2.89% vs 3.05%) but lower IFN score (HR+/HER2-: -0.56 vs -0.41 vs-0.35; HER2+: -0.53 vs -0.39 vs -0.30; TNBC: -0.55 vs -0.30 vs -0.28). Conclusions: BM in BC are more frequently associated with TNBC and Basal-like BC phenotypes than ECM or PTs. BM have a higher proportion of ESR1-mut than PTs, highlighting a possible association between endocrine resistance and development of BM. BM had lower IFN score and PDL-1 expression highlighting an immunosuppressed tumor microenvironment. [Table: see text]

Real-world outcomes and molecular correlates of incorporating immunotherapy (IO) and bevacizumab (Bev) in treating patients with peritoneal mesothelioma (PeM): A comparative analysis with pleural mesothelioma (PM).

e23300 Background: Nivolumab/Ipilimumab (IO) and Bev/Chemo are now considered standards of care for patients (pts) with advanced PM, based on randomized (CheckMate743 and MAPS) trials. By extrapolation, they are also endorsed by guidelines and used clinically in pts with PeM. Due to lack of any prospective data, we evaluated real-world effectiveness of these agents in PeM (vs PM) and investigated molecular correlates underlying their therapeutic impact. Methods: The study cohort was composed of mesothelioma pts (N = 723) who underwent DNA (592-gene panel or whole exome)/RNA (whole transcriptome) [PeM (221/224) and PM (493/499)] sequencing at Caris Life Sciences (Phoenix, AZ). Treatment and real-world survival (OS) were obtained from insurance claims and calculated from time of tissue collection to last contact. Hazard ratio (HR) was calculated using Cox proportional hazards model. Tumor microenvironment (TME) estimates were calculated from bulk RNA sequencing using QuanTIseq method. Significance was determined using Chi-square, Mann Whitney U and log-rank tests and corrected for multiple comparisons (q-value < 0.05) when applicable. Results: PeM (vs PM) had a higher proportion of females (46% vs 29%) and younger age (62 vs 73 years; both p < 0.001). 20% and 22% of PeM were treated with IO or Bev/Chemo, with similar utilization rates in PM of 22% and 24%, respectively. OS estimates and comparisons are shown in Table. Prevalence of mutations in key mesothelioma associated genes ( BAP1, NF2, TP53 and SETD2) was not significantly different between PeM and PM. NF2 (10.1 vs 51.3 m for wild type; p < 0.001) and TP53 (10.4 vs 46.6 m for wild type; p < 0.006) mutations were associated with worse OS in PeM but not in PM. Prevalence of high tumor mutation burden (≥10 mut/Mb: 1.0% vs 3.4%; q = 0.37) and PD-L1+ (18.7% vs 28.8%; q = 0.07) tumors was slightly lower in PeM than PM. Macrophage fractions, M2 were higher (1.5-fold) and M1 were lower (0.8-fold) in PeM (both q < 0.05) compared to PM. Conclusions: In this large real-world analysis, use of IO appears to be associated with improved outcomes in PeM, despite a potentially immunosuppressive TME. Notably, addition of Bev did not appear to improve outcomes in PeM, in contrast to PM. These results highlight variances in clinical utility/value of these therapies. Molecular mechanisms underlying these outcome differences warrant further investigation. [Table: see text]

Neoantigen-augmented iPSC cancer vaccine combined with radiotherapy promotes antitumor immunity in poorly immunogenic cancers

Although irradiated induced-pluripotent stem cells (iPSCs) as a prophylactic cancer vaccine elicit an antitumor immune response, the therapeutic efficacy of iPSC-based cancer vaccines is not promising due to their insufficient antigenicity and the immunosuppressive tumor microenvironment. Here, we found that neoantigen-engineered iPSC cancer vaccines can trigger neoantigen-specific T cell responses to eradicate cancer cells and increase the therapeutic efficacy of RT in poorly immunogenic colorectal cancer (CRC) and triple-negative breast cancer (TNBC). We generated neoantigen-augmented iPSCs (NA-iPSCs) by engineering AAV2 vector carrying murine neoantigens and evaluated their therapeutic efficacy in combination with radiotherapy. After administration of NA-iPSC cancer vaccine and radiotherapy, we found that ~60% of tumor-bearing mice achieved a complete response in microsatellite-stable CRC model. Furthermore, splenocytes from mice treated with NA-iPSC plus RT produced high levels of IFNγ secretion in response to neoantigens and had a greater cytotoxicity to cancer cells, suggesting that the NA-iPSC vaccine combined with radiotherapy elicited a superior neoantigen-specific T-cell response to eradicate cancer cells. The superior therapeutic efficacy of NA-iPSCs engineered by mouse TNBC neoantigens was also observed in the syngeneic immunocompetent TNBC mouse model. We found that the risk of spontaneous lung and liver metastasis was dramatically decreased by NA-iPSCs plus RT in the TNBC animal model. Altogether, these results indicated that autologous iPSC cancer vaccines engineered by neoantigens can elicit a high neoantigen-specific T-cell response, promote tumor regression, and reduce the risk of distant metastasis in combination with local radiotherapy.

Multifunctional bispecific nanovesicles targeting SLAMF7 trigger potent antitumor immunity.

The effectiveness of immune checkpoint inhibitor (ICI) therapy is hindered by the ineffective infiltration and functioning of cytotoxic T cells and the immunosuppressive tumor microenvironment (TME). Signaling lymphocytic activation molecule family member 7 (SLAMF7) is a pivotal co-stimulatory receptor thought to simultaneously trigger natural killer (NK)-cell, T-cell, and macrophage antitumor cytotoxicity. However, the potential of this collaborative immune stimulation in antitumor immunity for solid tumors is under-explored due to the exclusive expression of SLAMF7 by hematopoietic cells. Here, we report the development and characterization of multifunctional bispecific nanovesicles targeting SLAMF7 and Glypican-3-a hepatocellular carcinoma (HCC)-specific tumor antigen. We found that by effectively "decorating" the surface of solid tumors with SLAMF7, these nanovesicles directly induced potent and specific antitumor immunity and remodeled the immunosuppressive TME, sensitizing the tumors to programmed cell death protein 1 (PD-1) blockade. Our findings highlight the potential of SLAMF7-targeted multifunctional bispecific nanovesicles as an anticancer strategy with implications for designing next-generation targeted cancer therapies.

Qingfei mixture mitigates immunosuppression of tumor microenvironment in non-small cell lung cancer by blocking stat1/Ido1-mediated tryptophan-kynurenine pathway

Programmed death-1 (PD-1) acts as a T cell checkpoint and is important in controlling T cell exhaustion. Blocking the intercommunication across PD-1 and PD-L1 is promising for advanced lung cancer treatment. However, the response rate requires being strengthened. This study aimed to determine whether the combination treatment of Qingfei mixture (QFM) and PD-1 inhibitor could improve the sensitivity of monoclonal antibody by regulating STAT1/IDO1-mediated tryptophan (Trp)-kynurenine (Kyn) pathway. The in vivo imaging system, immunofluorescence, hematoxylin-eosin staining, TUNEL, flow cytometry, HPLC, and ELISA were used to estimate the anti-tumor effects in LLC-luc tumor-bearing C57BL/6 mice treated with QFM, PD-1 inhibitor, 2-NP (enhancer of STAT1 transcription), and FICZ (AhR agonist) alone or in combination. IFN-γ-mediated A549 and LLC cells were treated with QFM-containing serum and fludarabine (FLU, STAT1 inhibitor), and cell viability, apoptosis, and Kyn content were then evaluated using CCK-8 assays, flow cytometry, and HPLC assays, respectively. Additionally, the expressions of STAT1, IDO1, AhR, NFATc1, TRIP12, PD-1, and PD-L1 were measured in vivo and in vitro. We found QFM increased the anti-cancer actions of PD-1 inhibitors by increasing the CD8+IFNγ+ T cells infiltration and decreasing the ratio of Kyn/Trp. Besides, QFM-containing serum suppressed the proliferation and promoted apoptosis in A549 and LLC cells, meanwhile, FLU boosted the effects of QFM-containing serum. Moreover, the suppression of tumor growth in the combination therapy was attenuated in the mice receiving 2-NP or FICZ. The occurrence of the above results was accompanied by a decrease in STAT1, IDO1, AhR, PD-1, and PD-L1 expressions. Collectively, the findings suggested that QFM may increase the influences of PD-1 inhibitors at least partially by blocking the STAT1/IDO1-mediated tryptophan-kynurenine pathway in lung cancer.

Multi-model analysis of gallbladder cancer reveals the role of OxLDL-absorbing neutrophils in promoting liver invasion

BackgroundGallbladder cancer (GBC) is the most common and lethal malignancy of the biliary tract that lacks effective therapy. In many GBC cases, infiltration into adjacent organs or distant metastasis happened long before the diagnosis, especially the direct liver invasion, which is the most common and unfavorable way of spreading.MethodsSingle-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST), proteomics, and multiplexed immunohistochemistry (mIHC) were performed on GBC across multiple tumor stages to characterize the tumor microenvironment (TME), focusing specifically on the preferential enrichment of neutrophils in GBC liver invasion (GBC-LI).ResultsMulti-model Analysis reveals the immunosuppressive TME of GBC-LI that was characterized by the enrichment of neutrophils at the invasive front. We identified the context-dependent transcriptional states of neutrophils, with the Tumor-Modifying state being associated with oxidized low-density lipoprotein (oxLDL) metabolism. In vitro assays showed that the direct cell-cell contact between GBC cells and neutrophils led to the drastic increase in oxLDL uptake of neutrophils, which was primarily mediated by the elevated OLR1 on neutrophils. The oxLDL-absorbing neutrophils displayed a higher potential to promote tumor invasion while demonstrating lower cancer cytotoxicity. Finally, we identified a neutrophil-promoting niche at the invasive front of GBC-LI that constituted of KRT17+ GBC cells, neutrophils, and surrounding fibroblasts, which may help cultivate the oxLDL-absorbing neutrophils.ConclusionsOur study reveals the existence of a subset of pro-tumoral neutrophils with a unique ability to absorb oxLDL via OLR1, a phenomenon induced through cell-cell contact with KRT17+ GBC cells in GBC-LI.