Anti-tumor Immune Microenvironment Research Articles

Identification of microtubule-associated biomarker using machine learning methods in osteonecrosis of the femoral head and osteosarcoma

BackgroundThis study aims to explore the microtubule-associated gene signatures and molecular processes shared by osteonecrosis of the femoral head (ONFH) and osteosarcoma (OS). MethodsDatasets from the TARGET and GEO databases were subjected to bioinformatics analysis, including the functional enrichment analysis of genes shared by ONFH and OS. Prognostic genes were identified using univariate and multivariate Cox regression analyses to develop a risk score model for predicting overall survival and immune characteristics. Furthermore, LASSO and SVM-RFE algorithms identified biomarkers for ONFH, which were validated in OS. Function prediction, ceRNA network analysis, and gene–drug interaction network construction were subsequently conducted. Biomarker expression was then validated on clinical samples by using qPCR. ResultsA total of 14 microtubule-associated disease genes were detected in ONFH and OS. Subsequently, risk score model based on four genes was then created, revealing that patients with low-risk exhibited superior survival outcomes compared with those with high-risk. Notably, ONFH with low-risk profiles may manifest an antitumor immune microenvironment. Moreover, by utilizing LASSO and SVM-RFE algorithms, four diagnostic biomarkers were pinpointed, enabling effective discrimination between patients with ONFH and healthy individuals as well as between OS and normal tissues. Additionally, 21 drugs targeting these biomarkers were predicted, and a comprehensive ceRNA network comprising four mRNAs, 71 miRNAs, and 98 lncRNAs was established. The validation of biomarker expression in clinical samples through qPCR affirmed consistency with the results of bioinformatics analysis. ConclusionMicrotubule-associated genes may play pivotal roles in OS and ONFH. Additionally, a prognostic model was constructed, and four genes were identified as potential biomarkers and therapeutic targets for both diseases.

CXCR6 expression correlates with radiotherapy response and immune context in triple-negative breast cancer (Experimental Studies)

Background: The chemokine receptor CXCR6 is critical for sustained tumor control mediated by CD8+ cytotoxic T cells (CTLs) in tumors. Previous studies have shown that ionizing radiation induces an inflamed immune contexture by upregulating CXCR6. However, the clinical significance of CXCR6 expression in triple-negative breast cancer (TNBC) and its correlation with radiotherapy remains unknown. This study aimed to clarify the prognostic value of CXCR6 and its role in the breast tumor microenvironment (TME). Methods: The mRNA and protein expression of CXCR6 in human triple-negative breast cancer and their association with survival were analyzed. The role of CXCR6 in the immune context was investigated using a combination of single-cell RNA sequencing, general transcriptome sequencing data and fluorescence-based multiplex immunohistochemistry (mIHC) techniques. Results: Elevated CXCR6 expression correlated with better clinical outcomes and superior response to adjuvant radiotherapy and immunotherapy in TNBC. CXCR6 fostered an immunostimulatory microenvironment characterized by upregulated cytotoxic markers. We also found that CXCR6 plays a crucial role in regulating the differentiation of CD8+ T cells and the intercellular communication of immune cell subtypes, shaping the anti-tumor microenvironment. Conclusions: This study highlights the emerging role of CXCR6 in shaping the antitumor immune microenvironment, and targeting CXCR6 may be a promising strategy for improving the effectiveness of radiotherapy and immunotherapy in TNBC.

Glutaryl-CoA dehydrogenase suppresses tumor progression and shapes an anti-tumor microenvironment in hepatocellular carcinoma

Crotonylation, a crotonyl-CoA-based non-enzymatic protein translational modification, affects diverse biological processes, such as spermatogenesis, tissue injury, inflammation, and neuropsychiatric diseases. Crotonylation shows decreased in hepatocellular carcinomas (HCCs), but the mechanism remains unknown. In this study, we aim to describe the role of glutaryl-CoA dehydrogenase (GCDH) in tumor suppression. Three cohorts containing 40, 248 and 17 pairs of samples were used to evaluate the link between GCDH expression levels and the HCC clinical characteristics as well as anti-PD-1 response. Subcutaneous xenograft, orthotopic xenograft, Trp53Δhep/Δhep; MYC- as well as Ctnnboe; METoe- driven mouse models were adopted to validate GCDH effects on HCC suppression. GCDH depletion promoted HCC growth and metastasis, whereas its overexpression reversed these processes. As GCDH converts glutaryl-CoA to crotonyl-CoA to increase crotonylation levels, we performed lysine crotonylome analysis and identified the pentose phosphate pathway (PPP) and glycolysis-related proteins PGD, TKT, and ALDOC as GCDH-induced crotonylation targets. Crotonyl-bound targets showed allosteric effects that controlled their enzymatic activities, leading to decreases in ribose 5-phosphate and lactate production, further limiting the Warburg effect. PPP blockade also stimulated peroxidation, synergizing with senescent modulators to induce senescence in GCDHhigh cells. These cells induced the infiltration of immune cells by the senescence-associated secretory cell phenotype (SASP) to shape an anti-tumor immune microenvironment. Meanwhile, the GCDHlow population was sensitized to anti-programmed cell death protein 1 (PD-1) therapy. GCDH inhibits HCC progression via crotonylation-induced suppression of the PPP and glycolysis, resulting in HCC cell senescence. The senescent cell further shapes an anti-tumor microenvironment by SASP. The GCDHlow population is vulnerable to anti-PD-1 therapy because more PD-1+CD8+ T cells are exhibited in GCDHlow population. GCDH is a favorable prognostic indicator in liver, lung, and renal cancers. In addition, most of GCDH depletion-induced toxic metabolites originate from the liver, accumulate locally, and cannot cross the blood-brain barrier. Therefore, studies on the correlation between GCDH and liver cancer would contribute to discovering the initiation and progression of hepatocellular carcinoma, of which over 70% of patients occupied >2-fold GCDH downregulation. Given that the GCDHlow and GCDHhigh HCC population can be distinguished based on serum glucose and ammonia levels, it will be worthwhile to evaluate the curative effects of pro-senescent and immune-therapeutic strategies based on the expression levels of GCDH.

Abstract 5924: RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models

Abstract Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide, and the third most common cause of cancer-related deaths. For the 40% to 50% of HCC patients who are candidates for systemic therapies, the landscape has evolved significantly in the last 10 years, with most recently, immunotherapies such as combination of atezolizumab and bevacizumab emerging as the current first-line systemic therapy for patients with intermediate or advanced disease. However, there are no predictive biomarkers for patients with HCC to enable selection of those most likely to benefit from these therapies and this remains a disease with high unmet medical need. Activation of the WNT pathway via mutational events or indirect pathway activation is implicated in many cancers including up to 50% of HCC patient tumors. High expression of beta-catenin, encoded by the CTNNB1 gene, has been shown to correlate with decreased survival and more rapid disease progression in HCC patients. In addition, WNT/beta-catenin pathway activation has been shown to result in immune exclusion and resistance to immunotherapy in HCC. ALN-BCAT comprises a chemically modified siRNA encapsulated in a lipid nanoparticle formulation which generates robust and highly specific reductions of CTNNB1 mRNA, downstream biomarkers and tumor cell proliferation in in vitro model systems. Anti-tumor activity has been demonstrated in multiple in vivo orthotopic HCC mouse models, in both early and late treatment settings as measured by liver to body weight ratio. Reductions in tumor burden were orthogonally confirmed by histology analysis and shown to be associated with changes in CTNNB1-related biomarkers. Finally, ALN-BCAT treatment results in changes in the tumor microenvironment in a mouse syngeneic HCC model, with increases in CD4+ and CD8+ T cells and reductions in M2 macrophages, consistent with a shift to a more immune stimulatory anti-tumor microenvironment. Here, we demonstrate that ALN-BCAT provides a novel approach to the inhibition of the WNT pathway as a therapeutic approach for treatment of HCC patients and WNT pathway activated tumors. Citation Format: Tulin Dadali, Spencer Miller, Gaurav Saawant, Stephen Abbott, Mikyung Yu, Svetlana Morskaya, Brandon Lehrich, Satdarshan Monga, Gloria Lau, Martin Maier, Wendy Broom. RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models [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 5924.

Abstract 6554: Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Repression of the MHC-I and reduced tumor infiltration of cytotoxic T-lymphocytes is often associated with immunotherapy resistance in SCLC. Lurbinectedin is FDA-approved as a second-line treatment for SCLC and we previously showed lurbinectedin as an effective therapeutic strategy in SCLC preclinical models. There are ongoing clinical trials combining immune checkpoint blockade with lurbinectedin in ES-SCLC. However, there is no insight into the effect of lurbinectedin on the immune microenvironment in SCLC. In this study, we evaluated the effect of lurbinectedin on the immune microenvironment and the anti-tumor effect with or without PD-L1 blockade. Results: To determine the effect of lurbinectedin in SCLC models, we treated immunocompetent flank RPP (Rb1, Trp53, and p130) and RPM (Rb1, Trp53, and MYCT58A) tumor-bearing mice with lurbinectedin (0.2 mg/kg, 1/7days) and/or anti-PD-L1 (300µg, 1/7days) for 3 weeks. Mice treated with anti-PD-L1 alone showed no anti-tumor response, and single-agent lurbinectedin caused a delay in tumor growth. However, 6 of the 10 mice treated with the combination of lurbinectedin and anti-PD-L1, had a complete response (100% reduction) and the other 4 had 90% tumor regression. Tumors were resected and analyzed by multicolor flow cytometry for changes in tumor-infiltrating lymphocytes (TILs). Furthermore, analysis of immune cells infiltrating into the tumors post- treatment showed significant induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells in the combination treatment arm. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased, while immunosuppressive M2 type macrophages and MDSC cells were decreased. These effects are consistent with data showing that lurbinectedin treatment leads to an activation of the cGAS/STING pathway, type I/II interferons (IFNα/β), and pro-inflammatory chemokines (CCL5, CXCL10) in tumors. Interestingly, lurbinectedin treatment led to significant upregulation of mRNA and surface expression of MHC class-I genes (HLA-A/B/C) in vitro and in vivo. Finally, bulk RNA sequencing as well as RT-PCR of pre- and post-lurbinectedin treatment demonstrated an increase in DAMPs associated with immunogenic cell death. Conclusion: We provide the first mechanistic insight into the lurbinectedin-induced multimodal immune modulation in SCLC leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Since lurbinectedin is already approved as a second-line agent in SCLC, our preclinical data provide a strong rationale for combining this regimen with inhibitors of the PD-L1 pathway and also highlight the immune subsets that the combination targets. Citation Format: Subhamoy Chakraborty, Yazhini Mahendravarman, Utsav Sen, Avisek Banerjee, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer [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 6554.

Abstract 2915: Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice

Abstract STK11 mutations occurs in 15-20% of lung adenocarcinoma (LUAD) and is associated with KRAS mutations, poor survival and resistance to immune checkpoint inhibitors (ICI). We observed tumor cell lines and primary human STK11-mutant LUAD upregulates of expression of C3 and complement factor H (FH). Deletion of C3 in STK11 knock-out tumor cells resulted in reduced FH expression. FH is a co-factor of Factor I, which converts C3b to proteolytically inactive iC3b thereby limiting activation of the alternative pathway (AP) C3 convertase. FH binds to proteoaminoglycans on the cell membrane and protects cells from AP complement-dependent cytotoxicity. With increased C3 generation by STK11-mutant tumor cells, we reasoned that systemic or tumor-derived FH protects tumor cells from complement attack. We hypothesized that tumor-derived C3 would promote STK11-mutant LUAD growth in immune intact mice by promoting immune evasion and would drive ICI resistance. Based on the expected tumor-protective effects of FH, we also hypothesized that anti-FH antibodies would control tumor growth and render tumor more sensitive to ICI. We evaluated growth of subcutaneously administered CMT167-STK11KO with and without C3 deletion (CMT167-STK11KO is murine KRASG12V LUAD with CRISPR/Cas9 deletion of STK11). Deletion of C3 in CMT167-STK11KO (CMT167-STK11KO/C3KO) resulted in dramatic inhibition of tumor growth and in sensitivity to anti-PD-1 therapy in vivo. In contrast, deletion of C3 in STK11-KO tumors had no effect on tumor growth in nude mice or following CD8 depletion in WT mice. These findings support a role of tumor-derived C3 in evasion of T cell anti-tumor immunity and as an obstacle to anti-PD-1 therapy. GT103 is a therapeutic antibody that binds tumor cell-attached CFH, causes complement activation, kills tumor cells, initiates a shift from a pro- to an anti-tumor immune microenvironment, and is currently being evaluated in a Phase 1b-2 clinical trial in combination with pembrolizumab (NCT05617313). We observed that single agent GT103 or anti-PD-1 has limited efficacy in CMT167-STK11KO tumor in WT mice. However, the combination of GT103 and anti-PD-1 was very effective in limiting tumor growth. Our results support a role of tumor-derived C3 in suppressing CD8 T-cell immunity and in driving ICI resistance and that the addition of GT103 renders STK11-KO tumors sensitive to anti-PD-1 in mice. These studies establish rationale for evaluating this combination in patients with STK11-mutant LUAD. Citation Format: Sora Suzuki, Anm Nazmul Khan, Thejaswini Giridharan, Catrina Ting, Ryan Bushey, Elizabeth Gottlin, Michael Campa, Edward F. Patz, Edwin H. Yau, Brahm Segal. Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice [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 2915.

POLQ identifies a better response subset to immunotherapy in muscle-invasive bladder cancer with high PD-L1.

Though programmed cell death-ligand 1 (PD-L1) has been used in predicting the efficacy of immune checkpoint blockade (ICB), it is insufficient as a single biomarker. As a key effector of an intrinsically mutagenic microhomology-mediated end joining (MMEJ) pathway, DNA polymerase theta (POLQ) was overexpressed in various malignancies, whose expression might have an influence on genomic stability, therefore altering the sensitivity to chemotherapy and immunotherapy. A total of 1304 patients with muscle-invasive bladder cancer (MIBC) from six independent cohorts were included in this study. The Zhongshan Hospital (ZSHS) cohort (n = 134), The Cancer Genome Atlas (TCGA) cohort (n = 391), and the Neo-cohort (n = 148) were included for the investigation of chemotherapeutic response. The IMvigor210 cohort (n = 234) and the UNC-108 cohort (n = 89) were used for the assessment of immunotherapeutic response. In addition, the relationship between POLQ and the immune microenvironment was assessed, and GSE32894 (n = 308) was used only for the evaluation of the immune microenvironment. We identified POLQhigh PD-L1high patients could benefit more from immunotherapy and platinum-based chemotherapy. Further analysis revealed that high POLQ expression was linked to chromosome instability and higher tumor mutational burden (TMB), which might elicit the production of neoantigens. Further, high POLQ expression was associated with an active tumor immune microenvironment with abundant infiltration of immune effector cells and molecules. The study demonstrated that high POLQ expression was correlated with chromosome instability and antitumor immune microenvironment in MIBC, and the combination of POLQ and PD-L1 could be used as a superior companion biomarker for predicting the efficacy of immunotherapy.

An NFAT1-C3a-C3aR Positive Feedback Loop in Tumor-Associated Macrophages Promotes a Glioma Stem Cell Malignant Phenotype.

Extensive infiltration by tumor-associated macrophages (TAM) in combination with myeloid-derived suppressor cells constitute the immunosuppressive microenvironment and promote the malignant phenotype of gliomas. The aggressive mesenchymal (MES)-subtype glioma stem cells (GSC) are prominent in the immunosuppressive microenvironment of gliomas. However, the underlying immune-suppressive mechanisms are still unknown. The current study showed that the antitumor immune microenvironment was activated in glioma in Nfat1-/- mice, suggesting induction of the immune-suppressive microenvironment by nuclear factor of activated T cells-1 (NFAT1). In TAMs, NFAT1 could upregulate the transcriptional activity of complement 3 (C3) and increase the secretion of C3a, which could then bind to C3aR and promote M2-like macrophage polarization by activating TIM-3. Simultaneously, C3a/C3aR activated the Ca2+-NFAT1 pathway, forming a positive feedback loop for the M2-like polarization of TAMs, which further promoted the MES transition of GSCs. Finally, disruption of this feedback loop using a C3aR inhibitor significantly inhibited glioma growth both in vitro and in vivo. The current study demonstrated that a NFAT1-C3a-C3aR positive feedback loop induces M2-like TAMs and further promotes the malignant phenotype of GSCs, which might be the potential therapeutic target for glioma.

Human papillomavirus infection affects treatment outcomes and the immune microenvironment in patients with advanced penile squamous cell carcinoma receiving programmed cell death protein 1 inhibitor-based combination therapy.

Penile squamous cell carcinoma (PSCC) is a human papillomavirus (HPV)-associated malignancy. Immunotherapy is emerging as a potential treatment for advanced PSCC. In this study, the authors analyzed the association of HPV status with outcomes and the immune microenvironment in patients with advanced PSCC undergoing programmed cell death protein 1 (PD1) inhibitor-based combination therapy (PCT). HPV status was assessed using quantitative polymerase chain reaction in 87 patients with advanced PSCC treated with PCT. Objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) in the HPV+ and HPV- groups were compared. Additionally, bulk RNA sequencing was performed to investigate the potential impact of HPV on the immune microenvironment in advanced PSCC. Among patients receiving first-line PCT, ORR (91.7% vs. 64.6%, p=.014) and DCR (100.0% vs. 79.2%, p=.025) in the HPV+group were higher compared to the HPV- group. Kaplan-Meier curves demonstrated that the HPV+group exhibited superior PFS (p=.005) and OS (p=.004) for patients in the first-line setting. However, these advantages of HPV infection were not observed in multi-line PCT (p>.050). HPV status remained an independent prognostic factor for predicting better ORR (p=.024), PFS (p=.002), and OS (p=.020) in the multivariate analyses. Landmark analyses showed that the HPV-induced superiority of PFS occurred at an early stage (within 3months) and OS occurred at a relatively late stage (within 9months). Bioinformatic analyses identified potential immune-activated genes (GLDC, CYP4F12, etc.) and pathways (RAGE, PI3K/AKT, etc.), antitumor immune cell subtypes, and lower tumor immune dysfunction and exclusion scores in HPV+tissues. HPV infection may confer treatment efficacy and survival benefits in patients with advanced PSCC receiving first-line PCT because of the possible stimulation of the antitumor immune microenvironment. Human papillomavirus (HPV) infection may induce better objective response rate, progression-free survival (PFS), and overall survival (OS) for advanced penile squamous cell carcinoma (PSCC) patients receiving first-line programmed cell death protein 1 inhibitor-based combination therapy (PCT) instead of multi-line PCT. HPV infection-induced PFS advantage occurs at an early stage (within 3months) whereas OS superiority occurs at a relatively late stage (within 9months). Antitumor immune microenvironment could be stimulated by HPV infection in advanced PSCC tissues.

Nuclear-Targeted Nanostrategy Regulates Spatiotemporal Communication for Dual Antitumor Immunity.

Intercellular communication between tumor cells and immune cells regulates tumor progression including positive communication with immune activation and negative communication with immune escape. An increasing number of methods are employed to suppress the dominant negative communication in tumors such as PD-L1/PD-1. However, how to effectively improve positive communication is still a challenge. In this study, a nuclear-targeted photodynamic nanostrategy is developed to establish positive spatiotemporal communication, further activating dual antitumor immunity, namely innate and adaptative immunity. The mSiO2 -Ion@Ce6-NLS nanoparticles (NPs) are designed, whose surface is modified by ionic liquid silicon (Ion) and nuclear localization signal peptide (NLS: PKKKRKV), and their pores are loaded with the photosensitizer hydrogen chloride e6 (Ce6). Ion-modified NPs enhance intratumoral enrichment, and NLS-modified NPs exhibit nuclear-targeted characteristics to achieve nuclear-targeted photodynamic therapy (nPDT). mSiO2 -Ion@Ce6-NLS with nPDT facilitate the release of damaged double-stranded DNA from tumor cells to activate macrophages via stimulator of interferon gene signaling and induce the immunogenic cell death of tumor cells to activate dendritic cells via "eat me" signals, ultimately leading to the recruitment of CD8+ T-cells. This therapy effectively strengthens positive communication to reshape the dual antitumor immune microenvironment, further inducing long-term immune memory, and eventually inhibiting tumor growth and recurrence.

Autophagy-amplifying nanoparticles evoke immunogenic cell death combined with anti-PD-1/PD-L1 for residual tumors immunotherapy after RFA

Incomplete radiofrequency ablation (IRFA) triggers mild protective autophagy in residual tumor cells and results in an immunosuppressive microenvironment. This accelerates the recurrence of residual tumors and causes resistance to anti-PD-1/PDL1 therapy, which bringing a great clinical challenge in residual tumors immunotherapy. Mild autophagy activation can promote cancer cell survival while further amplification of autophagy contributes to immunogenic cell death (ICD). To this regard, we constructed active targeting zeolitic imidazolate framework-8 (ZIF-8) nanoparticles (NPs) loaded with STF62247 or both STF62247 and BMS202, namely STF62247@ZIF-8/PEG-FA (SZP) or STF62247-BMS202@ZIF-8/PEG-FA (SBZP) NPs. We found that SZP NPs inhibited proliferation and stimulated apoptosis of residual tumor cells exposed to sublethal heat stress in an autophagy-dependent manner. Further results discovered that SZP NPs could amplify autophagy in residual tumor cells and evoke their ICD, which dramatically boosted the maturation of dendritic cells (DCs). Through vaccination experiments, we found for the first time that vaccination with heat + SZP treatment could efficiently suppress the growth of new tumors and establish long-term immunological memory. Furthermore, SBZP NPs could remarkably promote the ICD of residual tumor cells, obviously activate the anti-tumor immune microenvironment, and significantly inhibit the growth of residual tumors. Thus, amplified autophagy coupled with anti-PD-1/PDL1 therapy is potentially a novel strategy for treating residual tumors after IRFA.Graphical

Ferroptosis in cancer immunity and immunotherapy: Multifaceted interplay and clinical implications

Ferroptosis is a type of cell death characterized by iron-dependent phospholipid peroxidation and reactive oxygen species overproduction. Ferroptosis induces immunogenic cell death and elicits anti-tumor immune responses, playing an important role in cancer immunotherapy. Ferroptosis suppression in cancer cells impairs its immunotherapeutic efficacy. To overcome this issue, ferroptosis inducers (FINs) have been combined with other cancer therapies to create an anti-tumor immune microenvironment. However, the ferroptosis-based crosstalk between immune and tumor cells is complex because oxidative products released by ferroptotic tumor cells impair the functions of anti-tumor immune cells, resulting in immunotherapeutic resistance. In the present article, we have reviewed ferroptosis in tumor and immune cells and summarized the crosstalk between ferroptotic tumor cells and the immune microenvironment. Based on the existing literature, we have further discussed future perspectives on opportunities for combining ferroptosis-targeted therapies with cancer immunotherapies.

Inhibition of CDC20 potentiates anti-tumor immunity through facilitating GSDME-mediated pyroptosis in prostate cancer

BackgroundIncreasing evidence suggests that immunotherapy, especially immune checkpoint inhibitors (ICIs), has the potential to facilitate long-term survival in various cancer besides prostate cancer. Emerging evidence indicated that pyroptosis, an immunogenic form of cell death, could trigger an anti-tumor immune microenvironment and enhance the effectiveness of immunotherapy. Nevertheless, the mechanism underlying the regulation of pyroptosis signaling in prostate cancer remains unclear.MethodsThe differential expression of human E3 ligases in prostate cancer was integratedly analyzed from five independent public datasets. Moreover, the immunohistochemistry analysis of a tissue microarray derived from prostate cancer patients confirmed the results from the bioinformatic analysis. Furthermore, prostate cancer cell lines were evaluated via the next-generation RNA sequencing to assess transcriptomic profile upon CDC20 depletion. Next, qRT-PCR, Western blotting, cycloheximide assay, immunoprecipitation, and ubiquitination assay were employed to explore the correlation and interaction between CDC20 and GSDME. Both immune-deficient and immune-competent murine models were utilized to examine the anti-tumor efficacy of CDC20 inhibition with or without the anti-PD1 antibodies, respectively. To analyze the immune microenvironment of the xenografts, the tumor tissues were examined by immunohistochemistry and flow cytometry.ResultsThe analysis of multiple prostate cancer cohorts suggested that CDC20 was the most significantly over-expressed E3 ligase. In addition, CDC20 exerted a negative regulatory effect on the pyroptosis pathway by targeting GSDME for ubiquitination-mediated proteolysis in a degron-dependent manner. Knockdown of CDC20 leads to increased GSDME abundance and a transition from apoptosis to pyroptosis in response to death signals. Furthermore, in our syngeneic murine models, we found that depletion of CDC20 significantly enhances the anti-tumor immunity by promoting the infiltration of CD8+ T lymphocytes dependent on the existence of GSDME, as well as reducing myeloid immune cells. More importantly, Apcin, a small molecular inhibitor that targets CDC20, exhibited synergistic effects with anti-PD1-based immunotherapy in murine models of prostate cancer.ConclusionsOverall, these findings provide new insights into the upstream regulation of GSDME-mediated pyroptosis by CDC20, which specifically interacts with GSDME and facilitates its ubiquitination in a degron-dependent manner. Importantly, our data highlight novel molecular pathways for targeting cellular pyroptosis and enhancing the effectiveness of anti-PD1-based immunotherapy.

Multi-omics analysis revealing a senescence-relevant lncRNAs signature for the assessment of response to immunotherapy for breast cancer.

Breast cancer (BRCA) is the most fatal malignancy of women. Immunotherapy has greatly improved the prognosis of advanced BRCA. Cellular senescence contributes to tumorigenesis and suppresses anti-cancer immunity. Identification of senescence-relevant long noncoding RNAs (SRlncRNAs) signature may benefit the predictions of prognosis and response to immunotherapy of BRCA. RNA-seq, mutation, and clinical data of BRCA were acquired from public databases. SRlncRNAs were screened using univariate Cox regression analysis. Consensus clustering classified BRCA patients into 2 clusters, and the differences of overall survival (OS) and immune status between the 2 clusters were analyzed by survival analysis, CIBERSORT, and ESITIMATE. The SRlncRNAs signature was constructed by least absolute shrinkage and selection operator (LASSO) regression analysis, and BRCA patients were divided into 2 risk groups. Enrichment analyses were performed to explore the cancer- and immunotherapy-relevant pathways. Transcriptome analysis was performed to investigate the differences of OS, immune infiltration, and ESITIMATE score of the 2 groups. Genome analysis was applied to investigate the differences of somatic mutation, tumor mutation burden (TMB) and microsatellite instability (MSI) between the 2 risk groups. A nomogram combined with calibration curves and decision curve analysis (DCA) was established for better clinical decision. Tumor Immune Dysfunction and Exclusion (TIDE) score and IMvigor-210 were applied for the predicting of response to immunotherapy. Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) and the Cancer Therapeutics Response Portal resource (CTRP) databases were used for drug susceptibility analysis. Ten prognostic SRlncRNAs were identified and BRCA patients were divided into 2 clusters. Cluster 1 had better OS with anti-tumor immune microenvironment. The high-risk BRCA had poorer OS in the Cancer Genome Atlas (TCGA) training cohort, which was also verified by TCGA validation cohort and GSE20685 validation cohort. Low-risk patients also had anti-tumor immune microenvironment. Genome analysis demonstrated that the high-risk group had significant higher TMB. High-risk BRCA were more susceptive to immunotherapy according to the TIDE score and IMvigor-210. Finally, drug susceptibility analysis showed that 6 compounds were sensitive to high-risk BRCA patients. We developed and verified an original SRlncRNAs signature by multi-omics analysis, which could serve as a prognosis and immunotherapy predictor for BRCA.

Lactate Efflux Inhibition by Syrosingopine/LOD Co-Loaded Nanozyme for Synergetic Self-Replenishing Catalytic Cancer Therapy and Immune Microenvironment Remodeling.

An effective systemic mechanism regulates tumor development and progression; thus, a rational design in a one-stone-two-birds strategy is meant for cancer treatment. Herein, a hollow Fe3 O4 catalytic nanozyme carrier co-loading lactate oxidase (LOD) and a clinically-used hypotensor syrosingopine (Syr) are developed and delivered for synergetic cancer treatment by augmented self-replenishing nanocatalytic reaction, integrated starvation therapy, and reactivating anti-tumor immune microenvironment. The synergetic bio-effects of this nanoplatform stemmed from the effective inhibition of lactate efflux through blocking the monocarboxylate transporters MCT1/MCT4 functions by the loaded Syr as a trigger. Sustainable production of hydrogen peroxide by catalyzation of the increasingly residual intracellular lactic acid by the co-delivered LOD and intracellular acidification enabled the augmented self-replenishing nanocatalytic reaction. Large amounts of produced reactive oxygen species (ROS) damaged mitochondria to inhibit oxidative phosphorylation as the substituted energy supply upon the hampered glycolysis pathway of tumor cells. Meanwhile, remodeling anti-tumor immune microenvironment is implemented by pH gradient reversal, promoting the release of proinflammatory cytokines, restored effector T and NK cells, increased M1-polarize tumor-associated macrophages, and restriction of regulatory T cells. Thus, the biocompatible nanozyme platform achieved the synergy of chemodynamic/immuno/starvation therapies. This proof-of-concept study represents a promising candidate nanoplatform for synergetic cancer treatment.

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth

BackgroundThough the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established.MethodsTo characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten−/−/Cxcr2−/− and NRasQ61R/INK4a−/−/Cxcr2−/− melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten−/− and NRasQ61R/INK4a−/− mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA).ResultsGenetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models.ConclusionsHere, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

Spatial distribution of tumor-infiltrating T cells indicated immune response status under chemoradiotherapy plus PD-1 blockade in esophageal cancer.

The spatial distribution of tumor-infiltrating T cells and its dynamics during chemoradiotherapy combined with PD-1 blockade is little known in esophageal squamous cell carcinoma (ESCC). We applied the multiplex immunofluorescence method to identify T cells (CD4+, CD8+ T cells, and their PD-1- or PD-1+ subsets) and myeloid-derived cells (CD11c+ dendritic cells, CD68+ macrophages, and their PD-L1+ subpopulations) in paired tumor biopsies (n = 36) collected at baseline and during combination (40 Gy of radiation) from a phase Ib trial (NCT03671265) of ESCC patients treated with first-line chemoradiotherapy plus anti-PD-1 antibody camrelizumab. We used the FoundationOne CDx assay to evaluate tumor mutational burden (TMB) in baseline tumor biopsies (n = 14). We dynamically assessed the nearest distance and proximity of T-cell subsets to tumor cells under combination and estimated the association between T-cell spatial distribution and combination outcome, myeloid-derived subsets, TMB, and patient baseline characteristics. We found that the tumor compartment had lower T-cell subsets than the stromal compartment but maintained a comparable level under combination. Both before and under combination, PD-1- T cells were located closer than PD-1+ T cells to tumor cells; T cells, dendritic cells, and macrophages showed the highest accumulation in the 5-10-μm distance. Higher CD4+ T cells in the tumor compartment and a shorter nearest distance of T-cell subsets at baseline predicted poor OS. Higher baseline CD4+ T cells, dendritic cells, and macrophages were associated with worse OS in less than 10-μm distance to tumor cells, but related with better OS in the farther distance. Higher on-treatment PD-1-positive-expressed CD4+ and CD8+ T cells within the 100-μm distance to tumor cells predicted longer OS. T cells, dendritic cells, and macrophages showed a positive spatial correlation. Both high TMB and smoking history were associated with a closer location of T cells to tumor cells at baseline. We firstly illustrated the T-cell spatial distribution in ESCC. Combining chemoradiotherapy with PD-1 blockade could improve the antitumor immune microenvironment, which benefits the treatment outcome. Further understanding the precision spatiality of tumor-infiltrating T cells would provide new evidence for the tumor immune microenvironment and for the combination treatment with immunotherapy.

Molecular cluster mining of high-grade serous ovarian cancer via multi-omics data analysis aids precise medicine.

HGSOC is a kind of gynecological cancer with high mortality and strong heterogeneity. The study used multi-omics and multiple algorithms to identify novel molecular subtypes, which can help patients obtain more personalized treatments. Firstly, the consensus clustering result was obtained using a consensus ensemble of ten classical clustering algorithms, based on mRNA, lncRNA, DNA methylation, and mutation data. The difference in signaling pathways was evaluated using the single-sample gene set enrichment analysis (ssGSEA). Meanwhile, the relationship between genetic alteration, response to immunotherapy, drug sensitivity, prognosis, and subtypes was further analyzed. Finally, the reliability of the new subtype was verified in three external datasets. Three molecular subtypes were identified. Immune desert subtype (CS1) had little enrichment in the immune microenvironment and metabolic pathways. Immune/non-stromal subtype (CS2) was enriched in the immune microenvironment and metabolism of polyamines. Immune/stromal subtype (CS3) not only enriched anti-tumor immune microenvironment characteristics but also enriched pro-tumor stroma characteristics, glycosaminoglycan metabolism, and sphingolipid metabolism. The CS2 had the best overall survival and the highest response rate to immunotherapy. The CS3 had the worst prognosis and the lowest response rate to immunotherapy but was more sensitive to PARP and VEGFR molecular-targeted therapy. The similar differences among three subtypes were successfully validated in three external cohorts. We used ten clustering algorithms to comprehensively analyze four types of omics data, identified three biologically significant subtypes of HGSOC patients, and provided personalized treatment recommendations for each subtype. Our findings provided novel views into the HGSOC subtypes and could provide potential clinical treatment strategies.

Pre-operative exercise therapy suppresses colorectal liver metastases by triggering anti-tumor trained immunity of Kupffer cells

Abstract Hepatic recurrence after surgical resection of colorectal liver metastasis is the major cause of treatment failure. Pre-operative exercise therapy (PEx) has been shown to improve outcomes from major abdominal surgeries. We demonstrate that PEx significantly attenuates liver ischemia/reperfusion (I/R) injury by inducing an anti-inflammatory phenotype in Kupffer cells (KCs). However, the mechanisms by which PEx prevents surgery-induced hepatic recurrence remain unclear. Results: Four weeks of treadmill running significantly reduces I/R-induced hepatic metastatic tumor growth in mice compared to sedentary controls. Depletion of KCs substantially decreases I/R-induced tumor progression, indicating a pro-tumor role of KCs. Our scRNA-seq data reveal that PEx causes dramatic transcriptomic changes in KCs. A clear induction of trained immunity with noticeably higher levels of anti-tumor cytokines IFN-γ and TNF-α is observed in PEx-KCs. With scATAC-seq, we find that PEx triggers the chromatin accessibilities profile changes in KCs, which is associated with the metabolic reprogramming of itaconate/IRG1 pathway. Furthermore, PEx significantly changes the transcriptomic profile of polymorphonuclear myeloid-derived suppressive cells (PMN-MDSCs) in the tumor microenvironment (TME) and decreases them from forming neutrophil extracellular traps (NETs), which promote tumor progression. We identify the cell-cell interaction between KCs and PMN-MDSCs in the TME is significantly enriched after PEx, suggesting that PEx may facilitate KCs to modulate PMN-MDSCs. In summary, PEx ameliorates surgical stress-induced pro-tumorigenic inflammatory responses and sustains an anti-tumor immune microenvironment in the liver. Supported by grants from NIH (R01GM137203 for Hai Huang, R01CA214865 for Allan Tsung)

Cancer Cell Membrane-Coated Gambogic Acid Nanoparticles for Effective Anticancer Vaccination by Activating Dendritic Cells.

Recent studies have shown that traditional Chinese medicine (TCM), such as gambogic acid (GA), is involved in the regulation of tumor immune microenvironment and can be combined with other anti-tumor treatment strategies. Here, we used GA as an adjuvant to construct a nano-vaccine to improve the anti-tumor immune response of colorectal cancer (CRC). We used a previously reported two-step emulsification method to obtain poly (lactic-co-glycolic acid) /GA nanoparticles (PLGA/GA NPs), and then CT26 colon cancer cell membrane (CCM) was used to obtain CCM-PLGA/GA NPs. This novel nano-vaccine, CCM-PLGA/GA NPs, was co-synthesized with GA as an adjuvant and neoantigen provided by CT26 CCM. We further confirmed the stability, tumor targeting, and cytotoxicity of CCM-PLGA/GA NPs.The regulatory effect on the tumor immune microenvironment, the anti-tumor efficacy, and the combined anti-tumor efficacy with anti-PD-1 monoclonal Antibodies (mAbs) of this novel nano-vaccine was also detected in vivo. We successfully constructed the CCM-PLGA/GA NPs. In vitro and in vivo tests showed low biological toxicity, as well as the high tumor-targeting ability of the CCM-PLGA/GA NPs. Besides, we revealed a remarkable effect of CCM-PLGA/GA NPs to activate the maturation of dendritic cells (DCs) and the formation of a positive anti-tumor immune microenvironment. This novel nano-vaccine constructed with GA as the adjuvant and CCM providing the tumor antigen can not only directly kill tumors by enhancing the ability of GA to target tumors, but also indirectly kill tumors by regulating tumor immune microenvironment, providing a new strategy for immunotherapy of CRC.