Cytotoxic Lymphocyte Infiltration Research Articles

Promotion of Triple Negative Breast Cancer Immunotherapy by Combining Bioactive Radicals with Immune Checkpoint Blockade.

Although immunotherapy has revolutionized clinical cancer treatment, the efficacy is limited due to the lack of tumor-associated antigens (TAAs) and the presence of compensatory immune checkpoints. To overcome the deficiency, a nano-system loaded with ozone and CD47 inhibitor RRx-001 is designed and synthesized. Upon irradiation, reactive oxygen species (ROS) generated from ozone reacts with nitric oxide (NO) metabolized from RRx-001 to form reactive nitrogen species (RNS), which presents a much stronger cell-killing ability than ROS. Molecular mechanism studies further reveal that RNS induce extensive immunogenic cell death (ICD). The released TAAs promote infiltration of cytotoxic T lymphocytes, which provides the basis for immune checkpoint blockade (ICB) therapy. Meanwhile, RRx-001 carried by the nanoparticles and the produced radicals repolarize M2-type tumor-associated macrophages (TAMs) into the anti-tumor M1-type, consequently reversing the immunosuppressive tumor microenvironment (TME). In a xenograft triple-negative breast cancer (TNBC) animal model, O3-001@lipo (liposome enwrapping O3 and RRx-001) plus irradiation shows a significant anti-tumor efficacy by improving cytotoxic lymphocyte infiltration and regulating immunosuppressive TME. In summary, the O3-001@lipo nano-system triggered by irradiation potently improves the efficacy of immunotherapy by introducing strong cytotoxic RNS, which not only enriches the toolbox of ICD inducer but also provides a strategy of treatment for immune deficient tumor. STATEMENT OF SIGNIFICANCE: Significance 1: The significance of the work with respect to the existing literature This study introduces a nano-system that leverages ozone and RRx-001 in the presence of X-ray irradiation to generate reactive nitrogen species, enhancing immunogenic cell death and promoting T-lymphocyte infiltration in triple-negative breast cancer, addressing a significant unmet need in the field. Significance 2: The scientific impact and interest to our readership The scientific contribution is the development of a clinically translatable nano-system that not only induces ICD but also reshapes the tumor microenvironment, which is expected to have a profound impact on the readership in pharmaceutics, material science, and nano-bio interaction, particularly for those interested in advanced immune therapy approaches.

Abstract C041: MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer

Abstract Patients with castration-resistant prostate cancer (CRPC) are generally unresponsive to tumor targeted and immunotherapies. Whether genetic alterations acquired during the evolution of CRPC impact immune and immunotherapy responses is largely unknown. Using our innovative electroporation-based mouse models, we generated distinct genetic subtypes of CRPC found in patients and uncovered unique immune microenvironments. Specifically, mouse and human prostate tumors with MYC amplification and p53 disruption had weak cytotoxic lymphocyte infiltration and an overall dismal prognosis. MYC and p53 cooperated to induce tumor intrinsic secretion of VEGF, which by signaling through VEGFR2 expressed on CD8+ T cells, could directly inhibit T cell activity. Targeting VEGF-VEGFR2 signaling in vivo led to CD8+ T cell-mediated tumor and metastasis growth suppression and significantly increased overall survival in MYC and p53 altered CPRC. VEGFR2 blockade also led to induction of PD-L1, and in combination with PD-L1 immune checkpoint blockade produced anti-tumor efficacy in multiple preclinical CRPC mouse models. Thus, our results identify a genetic mechanism of immune suppression through VEGF signaling in prostate cancer that can be targeted to reactivate immune and immunotherapy responses in an aggressive subtype of CRPC. Citation Format: Katherine Murphy. MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C041.

A Highly Tumor-Permeating DNA Nanoplatform for Efficient Remodeling of Immunosuppressive Tumor Microenvironments.

The immunosuppressive tumor microenvironment and limited intratumoral permeation have largely constrained the outcome of tumor therapy. Herein, we report a tailored DNA structure-based nanoplatform with striking tumor-penetrating capability for targeted remodeling of the immunosuppressive tumor microenvironment in vivo. In our design, chemo-immunomodulator (gemcitabine) can be precisely grafted on DNA sequences through a reactive oxygen species (ROS)-sensitive linker. After self-assembly, the gemcitabine-grafted DNA structure can site-specifically organize legumain-activatable melittin pro-peptide (promelittin) on each vertex for intratumoral delivery and further function as the template to load photosensitizers (methylene blue) for ROS production. The tailored DNA nanoplatform can achieve targeted accumulation, highly improved intratumoral permeation, and efficient immunogenic cell death of tumor cells by laser irradiation. Finally, the immunosuppressive tumor microenvironment can be successfully remodeled by reducing multi-type immunosuppressive cells and enhancing the infiltration of cytotoxic lymphocytes in the tumor. This rationally developed multifunctional DNA nanoplatform provides a new avenue for the development of tumor therapy.

A Highly Tumor‐Permeating DNA Nanoplatform for Efficient Remodeling of Immunosuppressive Tumor Microenvironments

AbstractThe immunosuppressive tumor microenvironment and limited intratumoral permeation have largely constrained the outcome of tumor therapy. Herein, we report a tailored DNA structure‐based nanoplatform with striking tumor‐penetrating capability for targeted remodeling of the immunosuppressive tumor microenvironment in vivo. In our design, chemo‐immunomodulator (gemcitabine) can be precisely grafted on DNA sequences through a reactive oxygen species (ROS)‐sensitive linker. After self‐assembly, the gemcitabine‐grafted DNA structure can site‐specifically organize legumain‐activatable melittin pro‐peptide (promelittin) on each vertex for intratumoral delivery and further function as the template to load photosensitizers (methylene blue) for ROS production. The tailored DNA nanoplatform can achieve targeted accumulation, highly improved intratumoral permeation, and efficient immunogenic cell death of tumor cells by laser irradiation. Finally, the immunosuppressive tumor microenvironment can be successfully remodeled by reducing multi‐type immunosuppressive cells and enhancing the infiltration of cytotoxic lymphocytes in the tumor. This rationally developed multifunctional DNA nanoplatform provides a new avenue for the development of tumor therapy.

Clinical analysis of 10 cases with subcutaneous panniculitis-like T-cell lymphoma and tissue AURKA expression.

Due to its rarity, subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is often misdiagnosed as benign panniculitis, and there are no standardized treatment guidelines for SPTCL. Aurora kinase A (AURKA) plays a regulatory role in both mitosis and meiosis. Cells treated with an AURKA inhibitor showed severe mitotic delay, which triggered apoptosis. Ten cases of SPTCL were collected in this study, and immunohistochemistry was performed to detect AURKA expression in the skin tissues of these cases. Control groups were set as follows: 1) 10 cases of inflammatory panniculitis; 2) 9 healthy individuals. Fisher's exact test was used to compare the positive rates of AURKA among various groups. An average onset age of 27.3 years was found in 10 SPTCL cases. Clinically, these patients primarily presented with multiple subcutaneous nodules on the trunk and lower extremities, accompanied by intermittent high fever. One case showed lymph node metastasis, while no other distant organ metastasis being observed in any case. Pathologically, there was an infiltration of a large number of atypical lymphocytes within the fat lobules, characterized as a cytotoxic type. AURKA stanning was positive in 6 out of 10 SPTCL cases, while no positive cases were found in the control groups. 1) SPTCL predominantly affects young individuals and can be identified by nodular erythema on the trunk, intermittent high fever, and infiltration of atypical cytotoxic lymphocytes within fat lobules. 2) For early-stage cases without metastasis, monotherapy with glucocorticoids or immunosuppressants such as cyclosporine can be considered. 3) High expression of AURKA in SPTCL tissues suggests that AURKA could be a potential biomarker for disease diagnosis, providing a theoretical basis for further targeted therapy.

Abstract 1424: Epigenetic activation of tumoral IFNγresponse and pyroptosis overcomes immunotherapy resistance in hepatocellular carcinoma

Abstract Background: Immune checkpoint blockade (ICB) therapies by antibodies have revolutionized the treatment paradigm for a variety of cancers. Although subsets of people exhibit durable responses, resistance and relapse are common in hepatocellular carcinoma (HCC). We therefore aim to delineate the mechanism underlying the failure of ICB therapy in HCC, and more importantly to identify ICB-sensitizing therapeutic approaches. Methods: ICB-resistant orthotopic-grafted murine models were established via a serial selection of HCC cells in ICB-treated mice. Integrated multi-omics analysis (single-cell RNA-seq, single-cell ATAC-seq and ChIP-seq) were applied to decode the mechanisms underlying the ICB resistance and an epigenetic-mediated ICB response. Results: We successfully established the ICB-resistant murine models of HCC, in which tumor intrinsic interferon-gamma (IFNγ) response was most significantly suppressed and characterized by a “cold” tumor microenvironment with decreased lymphocyte activation and intratumoral infiltration. On the other hand, single cell RNA-seq analysis of human HCC biopsies revealed that patients with HDAC1/2/3-high HCC exhibit poor survival upon ICB therapy. Notably, we found that a novel specific HDAC1/2/3 inhibitor CXD101 synergized with PD-(L)1 antibody to induce immunogenic cell death, eradicate tumor and prolong survival in our ICB-resistant mouse models, which was accompanied by enhanced intratumoral infiltration, activation and anti-tumor memory formation of cytotoxic lymphocytes. Mechanistically, CXD101 combined with PD-(L)1 inhibition synergistically reactivated tumor IFNγ pathway via enhancing chromatin accessibility of downstream genes associated with antigen presentation and lymphocytes recruitment. Moreover, the activated immune system induced tumor cell pyroptosis, further igniting antitumor immunity and ICB response. Conclusions: Our findings suggest that corruption of tumor intrinsic IFNγ signaling may confer ICB resistance upon ICB therapy, which can be rectified by class I HDAC inhibitor mediated IFNγ response and tumor cell pyroptosis. Based on these findings, we have commenced a Phase II clinical study of a new epigenetic immunotherapy (CXD101 plus anti-PD-1) for HCC patients resisting anti-PD-(L)1 alone. This project is supported by the CRF (C4045-18W), GRF (14115820), TRS (T11-706/18-N), Li Ka Shing Foundation, CUHK Strategic Seed Funding for Collaborative Research Scheme. We also acknowledge support (funding and study medications) by Celleron Therapeutics. Citation Format: Yalin Tu, Haoran Wu, Chengpeng Zhong, Zhewen Xiong, Jing Wang, Patrick Pak-Chun Wong, Weiqin Yang, Jiahuan Lu, Zhixian Liang, Shufen Chen, Lingyun Zhang, Siyun Chen, Jingying Zhou, Ka-Fai To, Joseph Jao-Yiu Sung, Stephen Lam Chan, David Kerr, Nick La Thangue, Alfred Sze-Lok Cheng. Epigenetic activation of tumoral IFNγresponse and pyroptosis overcomes immunotherapy resistance in hepatocellular carcinoma [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 1424.

Abstract 4381: Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC

Abstract Background: Kindlins are a small gene family of FERM domain-containing adaptor proteins that function as essential drivers of integrin activation. Kindlin-2 (K2) is the most widely expressed member of the Kindlin family; its homozygous deletion in mice is embryonically lethal, K2 expression is also dysregulated in several human cancers, including the breast. We established K2 as a major driver of TNBC tumors progression and metastasis through the regulation of several hallmarks of cancer. A well-established pathway whereby K2 regulates TNBC oncogenic behavior is through the regulation of integrin inside-out signaling by directly binding to the cytoplasmic tail of integrin β subunit. Interestingly, our recent studies found K2 to play a major role in activating the TGF-β-mediated regulation of the CSF1/EGF signaling through macrophage polarization to the M2 tumorigenic state and their increased tumor infiltration, as well as inhibiting tumor infiltration of cytotoxic lymphocytes, therefore, driving tumor immune evasion. Here we present novel findings involving K2 in the stabilization of the β1-Integrin:TβRI complexes by establishing a physical bridge that links β1-Integrin to TβRI. Loss of K2 results in the degradation of this protein complex and therefore, the inhibition of the oncogenic pathways downstream of these two major regulators of several hallmarks of cancer. Methods: We applied a combination of in vitro assays; CRISPR/Cas9 gene editing, cell migration, 3D tumor-sphere, solid binding, co-immunoprecipitation, cell adhesion and spreading, as well as western blot and flow cytometry analyses with MDA-MB-231 and 4T1 TNBC cell lines. We also used preclinical in vivo mouse models of TNBC tumor progression and metastasis to support or investigations. Results: We found that the K2/β1-Integrin direct interaction is mediated through the C-terminal F3 domain of K2, while the K2/TβRI direct interaction is mediated through the F2 domain of K2. Disruption of this bridge, via CRISPR/Cas9-mediated knockout of K2, leads to β1-Integrin and TβRI degradation, and inhibition of the oncogenic pathways downstream of both β1-Integrin and TβRI, and inhibited tumor growth and metastasis in levels comparable to loss of expression of either β1-Integrin and TβRI. Treatment of the K2-deficent cells with the proteasome inhibitor MG-132 restored expression of both β1-Integrin and TβRI, confirming that K2 is required for the stabilization of the β1-integrin/TβR1 complexes at the protein level. Rescue of K2 expression in the K2-KO cells restored their oncogenic activities both in vitro and in vivo. Conclusion: We identified K2 to play a novel and major in the stabilization of the β1-integrin/TβR1 complexes and the regulation of their downstream oncogenic signaling. These studies have a significant translational impact as they may identify new therapeutically targeted pathways that are desperately needed for the treatment of TNBC tumors. Citation Format: Neelum A. Yousaf Zai, Lamyae El Khalki, Wei Wang, Justin Szpendyk, Khalid Sossey-Alaoui. Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC [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 4381.

Abstract 162: Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer

Abstract Background: Immune checkpoint blockade (ICB), which unleashes potent CD8+ T cell-based anti-tumor responses, has emerged as a promising therapeutic option for several cancer types. However, response rates stay limited in gastric cancer (GC) patients, indicating the necessity of combinational strategies to overcome other immunosuppressive factors. Fatty acid synthesis (FAS) is an indispensable metabolic pathway proven to support cancer cell survival. The rate-limiting enzyme of FAS, FASN, is upregulated in several cancers and could contribute to cancer immune escape. Though some investigations propose that specific gene alterations can cause FASN upregulation under the GC setting, the immunoregulatory role and therapeutic value of FASN in the tumor microenvironment (TME) of GC have not been fully elucidated. Methods: FASN expression was measured by Western Blotting and flow cytometry. Bioinformatic analysis was conducted via an online website (www.camoip.net). The syngeneic GC mouse model was established by subcutaneous inoculation of MFC cells in 615-line mice, treated by vehicles, FASN inhibitor (FASNi), anti-PD-1 antibodies, or FASNi+anti-PD-1 antibodies. TAM and CD8+ T cell depletion was implemented by anti-CSF1R and anti-CD8α antibodies. Bone marrow-derived macrophages (BMDMs) were extracted from the femurs of 615-line mice, induced by M-CSF and stimulated by LPS or IL-4. The proportions and functional status of immune cells were identified by flow cytometry. Results: FASN was upregulated in GC tissues and GC human cell lines. Bioinformatic analyses demonstrated that FASN expression was related to poor infiltration of cytotoxic lymphocytes, as well as the downregulation of pathways related to anti-cancer immune responses. FASNi inhibited tumor growth in a syngeneic GC mouse model, with elevated PD-1 and IFN-γ expression in CD8+ T cells and increased NK cell infiltration, suggesting the enhancement in anti-cancer cytotoxicity. Meanwhile, FASNi induced a shift from M2-like to M1-like in tumor-associated macrophages (TAM). We detected that M2-like TAM and CD8+ T cells harbored higher FASN expression than other immune cells in GC mouse tissues, and tumor-suppressing effects of FASN inhibition could be reversed by TAM and CD8+ T cell depletion, which corroborated the key role of TAM and CD8+ T cells in shaping FASN-mediated tumor regulation. FASNi could also repolarize BMDMs from M1-like to M2-like in vitro. Finally, FASNi exhibited synergistic tumor control with anti-PD-1 therapy in GC and melanoma mouse models. Conclusion: This work sheds light on the impact of FASN on TME modulation of GC. We proved that FASNi could repolarize M2-like TAM to M1 phenotypes and promote cytotoxic lymphocyte-mediated anti-tumor immunity. Our results propose FASN inhibition as a potential therapeutic strategy to sensitize ICB therapy in GC. Citation Format: Hanbing Wang, Tao Shi, Yue Wang, Yuting Luo, Yunfeng Pan, Xiaoyu Zhou, Yue Zhang, Baorui Liu, Jia Wei. Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric 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 162.

Abstract 3923: Targeting immunosuppressive TREM2+tumor associated macrophages in prostate cancer

Abstract With over 250,000 new cases annually, prostate cancer (PCa) is amongst the most diagnosed cancers in the United States. Despite a near-perfect 5-year survival rate in patients with localized disease with androgen deprivation therapy (ADT), about 10-20% of patients on ADT eventually develop castration-resistant prostate cancer (CRPC). Immunotherapies are typically ineffective in treating these treatment-resistant patients due to the abundance of immunosuppressive tumor-associated macrophages (TAMs) and lack of infiltrating lymphocytes. We propose to target these immunosuppressive macrophages through the triggering receptor expressed on myeloid cells 2 (TREM2), which has been identified as a major mediator of suppressive macrophage function but has not been studied in the context of PCa and CRPC. For this study, we analyzed publicly available RNA-seq datasets to query for TREM2 expression amongst patients with a range of prostate cancer disease states and cell types, measured tumor growth kinetics in two subcutaneous models of prostate cancer, and compared tumor immune infiltrates by flow cytometry. Our analysis of the TCGA prostate adenocarcinoma (PRAD) and the Stand Up to Cancer/Prostate Cancer Foundation (SU2C/PCF) datasets implicate TREM2 expression with prostate cancer progression, by Gleason score and progression-free status, and with worse overall survival of patients with metastatic CRPC (mCRPC). Manual curation and analysis of 10 single-cell RNA-seq datasets with a total of 84 patients, encompassing primary PCa, CRPC, and mCRPC indicate that TREM2 is highly and specifically expressed on TAMs in prostate cancer, with macrophages from patient-matched tumor samples expressing higher TREM2 compared to normal adjacent tissue. B6CaP tumors, with a luminal cytokeratin profile, high AR expression, and high Myc expression, show slower tumor growth in Trem2−/− mice compared to tumors in Trem2wt mice. On the other hand, RM1 tumors show no difference in growth kinetics on Trem2−/− mice. A major difference between the B6CaP and RM1 tumor microenvironments is the level of macrophage infiltration, where only 2-5% of immune cells in RM1 tumors are macrophages, compared to the 40-50% in B6CaP tumors. RM1 cells co-implanted with Trem2−/− macrophages show slower tumor growth, compared to tumor cells co-implanted with Trem2wt macrophages, confirming the role of Trem2-expressing macrophages in mediating tumor growth. Consistent with previous reports of suppressive Trem2 signaling, we also observed higher levels of inflammatory cytokine production in ex vivo stimulated Trem2−/− TAMs. By targeting TREM2 in prostate cancer, we find that TAMs are less suppressive, more inflammatory, and permit cytotoxic lymphocyte infiltration to enhance anti-tumor responses by synergizing with chemo and immune checkpoint therapy. Citation Format: Alex J. Lee, Kenneth Adusei, Monali Praharaj, Fan Shen, Xiaoxu Wang, Kaavya Bhatia, Thomas Nirschl, Jelani C. Zarif. Targeting immunosuppressive TREM2+tumor associated macrophages in prostate 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 3923.

RHBDF1 deficiency suppresses melanoma glycolysis and enhances efficacy of immunotherapy by facilitating glucose-6-phosphate isomerase degradation via TRIM32

Melanoma is a dangerous form of skin cancer, making it important to investigate new mechanisms and approaches to enhance the effectiveness of treatment. Here, we establish a positive correlation between the human rhomboid family-1 (RHBDF1) protein and melanoma malignancy. We demonstrate that the melanoma RHBDF1 decrease dramatically inhibits tumor growth and the development of lung metastases, which may be related to the impaired glycolysis. We show that RHBDF1 function is essential to the maintenance of high levels of glycolytic enzymes, especially glucose-6-phosphate isomerase (GPI). Additionally, we discover that the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) mediates the K27/K63-linked ubiquitination of GPI and the ensuing lysosomal degradation process. We prove that the multi-transmembrane domain of RHBDF1 is in competition with GPI, preventing the latter from interacting with NCL1-HT2A-LIN41 (NHL) domain of TRIM32. We also note that the mouse RHBDF1's R747 and Y799 are crucial for competitive binding and GPI protection. Artificially silencing the Rhbdf1 gene in a mouse melanoma model results in declined lactic acid levels, elevated cytotoxic lymphocyte infiltration, and improved tumor responsiveness to immunotherapy. These results provide credence to the hypothesis that RHBDF1 plays a significant role in melanoma regulation and suggest that blocking RHBDF1 may be an efficient technique for reestablishing the tumor immune microenvironment (TIME) in melanoma and halting its progression.

Nanodrug Augmenting Antitumor Immunity for Enhanced TNBC Therapy via Pyroptosis and cGAS-STING Activation.

Pyroptosis is a proinflammatory form of programmed cell death that results in the release of cellular contents and activation of immune responses. However, GSDME (a pyroptosis-executed protein) is suppressed in many cancers. Herein, we constructed a nanoliposome (GM@LR) for codelivering the GSDME-expressing plasmid and manganese carbonyl (MnCO) into TNBC cells. MnCO generated Mn2+ and carbon monoxide (CO) in the presence of H2O2. The CO-activated caspase-3, which cleaved the expressed GSDME, converting apoptosis to pyroptosis in 4T1 cells. In addition, Mn2+ promoted maturation of dendritic cells (DCs) by the activation of STING signaling pathway. The increased proportion of intratumoral mature DCs brought about massive infiltration of cytotoxic lymphocytes, leading to a robust immune response. Besides, Mn2+ could be applied for magnetic resonance imaging (MRI)-guided metastasis detection. Taken together, our study showed that GM@LR nanodrug could effectively inhibit tumor growth via pyroptosis and STING activation combined immunotherapy.

BMP9-induced vascular normalisation improves the efficacy of immunotherapy against hepatitis B virus-associated hepatocellular carcinoma.

In the past decade, the field of tumour immunotherapy has made a great progress. However, the efficacy of immune checkpoint blocking (ICB) in the treatment of hepatocellular carcinoma (HCC) remains limited. Cytotoxic lymphocyte trafficking into tumours is critical for the success of ICB. Therefore, additional strategies that increase cytotoxic lymphocyte trafficking into tumours are urgently needed to improve patient immune responses. Paired adjacent tissue and cancerous lesions with HBV-associated HCC were subjected to RNA-seq analysis. Bone morphogenetic protein (BMP9), which reflects vessel normalisation, was identified through Cytoscape software, clinical specimens and Gene Expression Omnibus (GEO) datasets for HCC. The functional effects and mechanism of BMP9 on the tumour vasculature were evaluated in cells and animals. An ultrasound-targeted microbubble destruction (UTMD)-mediated BMP9 delivery strategy was used to normalise the vasculature and evaluate therapeutic efficacy mediated by cytotoxic lymphocytes (NK cells) in combination with a PD-L1 antibody in human cancer xenografts of immune-deficient mice. We discovered that hepatitis B virus (HBV) infection-induced downregulation of BMP9 expression correlated with a poor prognosis and pathological vascular abnormalities in patients with HCC. BMP9 overexpression in HBV-infected HCC cells promoted intra-tumoural cytotoxic lymphocyte infiltration via vascular normalisation by inhibiting the Rho-ROCK-myosin light chain (MLC) signalling cascade, resulting in enhanced efficacy of immunotherapy. Furthermore, UTMD-mediated BMP9 delivery restored the anti-tumour function of cytotoxic lymphocytes (NK cells) and showed therapeutic efficacy in combination with a PD-L1 antibody in human cancer xenografts of immune-deficient mice. HBV-induced BMP9 downregulation causes vascular abnormalities that inhibit intra-tumoural cytotoxic lymphocyte infiltration, providing a rationale for developing and combining immunotherapy with BMP9-based therapy to treat HBV-associated HCC.

Abstract 1831: A long-acting and CD122-enhanced IL-2 analog, HM16390, synergizes with immune checkpoint inhibitor by remodeling an immune cell profile in tumor microenvironment

Abstract Immune checkpoint inhibitors (CPI) are widely used for cancer immunotherapy. However, the response to CPIs is dependent on phenotype of tumor microenvironment (TME). A cold tumor known as immune-excluded or -desert has shown poor response to CPIs due to an absence of effector T cells in TME. IL-2 analog, which is immune stimulator and able to recruit cancer-fighting cells into TME, may be promising therapeutic partner for overcoming a limitation of CPIs. Previously, HM16390, a long-acting CD-122-enhanced IL-2 analog, exerted dose-dependent anti-tumor activity in low immunogenic B16F10 melanoma mice. Here, we further investigated the immune cells composition in TME following HM16390 treatment and synergistic anti-tumor effect after combination with CPI. B16F10 mice were sacrificed at days 1, 3, and 8 following single subcutaneous administration of HM16390 or 5 consecutive daily administrations of aldesleukin. According to the result, HM16390 transiently increased peripheral cytokines (IFNγ and TNFα) more than aldesleukin. In line with this, we observed that the tumor-infiltrating NK/Treg ratio was significantly increased approximately 19 by treatment of HM16390 while showing 4.2 in aldesleukin treated group at Day 3. Furthermore, tumor-infiltrating CD8+/Treg ratio was also upregulated approximately 74 by treatment of HM16390 while only showing 6.1 in aldesleukin treated group at Day 8. Significantly increased pro-inflammatory molecules such as GrzB and IFNγ were also observed in HM16390 treated group compared to aldesleukin (p<0.01 and p<0.001, respectively). Next, we investigated synergistic anti-tumor effects in combination with CPI. B16F10 mice were repeatedly given HM16390 once a week or aldesleukin 5 consecutive days per week with or without mouse anti-PD1. After four weeks treatment, 25% of B16F10 mice showed complete response by treatment of HM16390, and significantly increased up to 88% by combination with anti-PD1. However, none of the mice showed complete response by treatment of aldesleukin/anti-PD1 combo. In conclusion, HM16390 effectively induced tumor growth inhibition through the activation and tumor infiltration of cytotoxic lymphocytes. This favorable immune alteration in tumor elicited a TME remodeling in which CPI could sufficiently respond. Citation Format: Jaehyuk Choi, Jinyoung Kim, Yu Yon Kim, Seongju Jeong, Sungmin Bae, Daejin Kim, In Young Choi. A long-acting and CD122-enhanced IL-2 analog, HM16390, synergizes with immune checkpoint inhibitor by remodeling an immune cell profile in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1831.

CX3CR1 deficiency-induced TIL tumor restriction as a novel addition for CAR-T design in solid malignancies

Advances in the understanding of the tumor microenvironment have led to development of immunotherapeutic strategies, such as chimeric antigen receptor Tcells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors invivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated Tcells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.

Heterogeneity of tumor immune microenvironment of EGFR/ALK-positive tumors versus EGFR/ALK-negative tumors in resected brain metastases from lung adenocarcinoma

BackgroundPrevious studies found that lung adenocarcinomas (LUAD) with EGFR-positive and ALK-positive were less responsive to immunotherapy, which may be associated with a suppressive tumor immune microenvironment (TIME). Given the discordance...

A Novel Tri-Functional Liposome Re-Educates "Cold Tumor" and Abrogates Tumor Growth by Synergizing Autophagy Inhibition and PD-L1 Blockade.

Immunotherapy has been regarded as a breakthrough in cancer treatment and achieved great success. However, the poor response rate is still a formidable challenge of current immunotherapies, especially in solid tumors without sufficient infiltration of immune cells, also known as "cold tumor." SAR405is a highly specific VPS34inhibitor and has been suggested as a potential approach converting "cold tumor" into "hot tumor" by inhibiting autophagy. In this study, a tri-functional doxorubicin (DOX) plus SAR405liposome system is established and further modified with a novel anti-PD-L1peptide JY4for targeted delivery (DOX-SAR-JY4LIPO ). The data here demonstrate that in a lung cancer xenograft mouse model, by facilitating the tumoral enrichment of both SAR405and DOX, DOX-SAR-JY4LIPO effectively increases the infiltration of cytotoxic lymphocytes in the tumor by synergizing DOX-induced immunogenic cell death (ICD) and SAR405-mediated upregulation of chemokines including CCL5and CXCL10. As results, DOX-SAR-JY4LIPO significantly inhibits tumor growth, metastasis, and resurrection by re-educating immunosuppressive tumor microenvironment. In conclusion, this study not only proves the concept of inhibiting autophagy for better immune infiltration in the tumor but also presents a novel tri-functional liposomal system that overcomes the deficiencies of current therapies and holds great promise in cancer immunotherapy.

Targeting CD47 enhanced the antitumor immunity of PD-L1 blockade in B-cell lymphoma.

Background: Only a subset of B-cell lymphoma (BCL) patients can benefit from immune checkpoint inhibitors targeting PD-1/PD-L1. Materials & methods: In the A20 model, SIRPα-Fc and anti-PD-L1 were employed to target CD47 and PD-L1 simultaneously. Flow cytometry, immunofluorescence and quantitative polymerase chain reactionwere used to unravel the potential mechanisms. Results: Simultaneously targeting CD47 and PD-L1 activated CD8+ T cells with an increased release of effector molecules. Furthermore, infiltration of F4/80+iNOS+ M1 macrophages was enhanced by the dual therapy. Conclusion: Anti-CD47 therapy could sensitize BCL tumors to anti-PD-L1 therapy in a CD8+ T-cell- and M1-macrophage-dependent manner bypromoting cytotoxic lymphocyte infiltration, which mayprovide a potential strategy for BCL treatment by simultaneously targeting CD47 and PD-L1.

Cannabinoid receptor 2 plays a pro-tumorigenic role in non-small cell lung cancer by limiting anti-tumor activity of CD8+ T and NK cells.

Cannabinoid (CB) receptors (CB1 and CB2) are expressed on cancer cells and their expression influences carcinogenesis in various tumor entities. Cells of the tumor microenvironment (TME) also express CB receptors, however, their role in tumor development is still unclear. We, therefore, investigated the role of TME-derived CB1 and CB2 receptors in a model of non-small cell lung cancer (NSCLC). Leukocytes in the TME of mouse and human NSCLC express CB receptors, with CB2 showing higher expression than CB1. In the tumor model, using CB1- (CB1 -/-) and CB2-knockout (CB2 -/-) mice, only deficiency of CB2, but not of CB1, resulted in reduction of tumor burden vs. wild type (WT) littermates. This was accompanied by increased accumulation and tumoricidal activity of CD8+ T and natural killer cells, as well as increased expression of programmed death-1 (PD-1) and its ligand on lymphoid and myeloid cells, respectively. CB2 -/- mice responded significantly better to anti-PD-1 therapy than WT mice. The treatment further increased infiltration of cytotoxic lymphocytes into the TME of CB2 -/- mice. Our findings demonstrate that TME-derived CB2 dictates the immune cell recruitment into tumors and the responsiveness to anti-PD-1 therapy in a model of NSCLC. CB2 could serve as an adjuvant target for immunotherapy.

Flow cytometry-assisted quantification of immune cells infiltrating irradiated tumors in mice.

The immunomodulatory properties of local hypofractionated radiotherapy are known to promote the generation of anti-tumor immune responses. Such responses are largely due to the infiltration of cytotoxic lymphocytes (TILs) into the tumors that are able to destroy malignant lesions. In this context, characterizing the tumor immune microenvironment following radiotherapy is crucial for the study of its mechanism of action. Flow cytometry-based analyses are frequently used to elucidate changes in the tumor immune microenvironment. The use of a fluorochrome-conjugated antibody panel is currently a standard technique to assess the number and phenotype of immune cell populations infiltrating the tumors. Here, we describe a method to isolate and quantify TILs based on flow-cytometry in mammary carcinoma-bearing mice that undergo a local hypofractionated radiotherapy regimen consisting of 3 consecutive doses of 8Gy. With some adaptations, this protocol can be successfully applied to a diverse range of transplantable and inducible solid mouse tumors of different origins.

ALKBH5 promotes PD-L1-mediated immune escape through m6A modification of ZDHHC3 in glioma

N6-methylation of adenosine (m6A) is one of the most frequent chemical modifications in eukaryotic RNAs and plays a vital role in tumorigenesis and progression. Recently, emerging studies have shown that m6A modification by ALKBH5 was associated with immunotherapy response in various types of cancer. However, whether m6A demethylases ALKBH5 participate in regulating the tumor immune microenvironment and the efficacy of immunotherapy in glioblastoma remain unknown. Here, we found that deletion of ALKBH5 significantly inhibited the growth of glioma allografts, rescued the antitumoral immune response, and increased cytotoxic lymphocyte infiltration and proinflammatory cytokines in CSF while significantly suppressing PD-L1 protein expression. m6A-methylated RNA immunoprecipitation sequencing and RNA sequencing identify ZDDHC3 as the direct target of ALKBH5. Mechanically, ALKBH5 deficiency impairs the YTHDF2-mediated stability of ZDHHC3 mRNA, thereby suppressing PD-L1 expression by accelerating PD-L1 degradation in glioma. In addition, genetic deletion or pharmacological inhibition of ALKBH5 with IOX1 enhances the therapeutic efficacy of anti-PD-1 treatment in preclinical mice models. These data suggest that the combination of anti-PD-1 therapy and ALKBH5 inhibition may be a promising treatment strategy in glioma.