Cells In Tumor-draining Lymph Nodes Research Articles

Fatty Acid Oxidation Supports Lymph Node Metastasis of Cervical Cancer via Acetyl-CoA-Mediated Stemness.

Accumulating evidence indicates that metabolic reprogramming of cancer cells supports the energy and metabolic demands during tumor metastasis. However, the metabolic alterations underlying lymph node metastasis (LNM) of cervical cancer (CCa) have not been well recognized. In the present study, it is found that lymphatic metastatic CCa cells have reduced dependency on glucose and glycolysis but increased fatty acid oxidation (FAO). Inhibition of carnitine palmitoyl transferase 1A (CPT1A) significantly compromises palmitate-induced cell stemness. Mechanistically, FAO-derived acetyl-CoA enhances H3K27 acetylation (H3K27Ac) modification level in the promoter of stemness genes, increasing stemness and nodal metastasis in the lipid-rich nodal environment. Genetic and pharmacological loss of CPT1A function markedly suppresses the metastatic colonization of CCa cells in tumor-draining lymph nodes. Together, these findings propose an effective method of cancer therapy by targeting FAO in patients with CCa and lymph node metastasis.

Engineered lactococcus lactis intrapleural therapy promotes regression of malignant pleural effusion by enhancing antitumor immunity

Intrapleural immunotherapies have emerged as a prominent field in treating malignant pleural effusion (MPE). Among these, bacteria-based intrapleural therapy has exerted an anti-MPE effect by immuno-stimulating or cytotoxic properties. We previously engineered a probiotic Lactococcus lactis (FOLactis) expressing a fusion protein of Fms-like tyrosine kinase 3 and co-stimulator OX40 ligands. FOLactis activates tumor antigen-specific immune responses and displays systemic antitumor efficacy via intratumoral delivery. However, no available lesions exist in the pleural cavity of patients with MPE for intratumoral administration. Therefore, we further optimize FOLactis to treat MPE through intrapleural injection. Intrapleural administration of FOLactis (I-Pl FOLactis) not only distinctly suppresses MPE and pleural tumor nodules, but also significantly extends noticeable survival in MPE-bearing murine models. The proportion of CD103+ dendritic cells (DCs) in tumor-draining lymph nodes increases three-fold in FOLactis group, compared to the wild-type bacteria group. The enhanced DCs recruitment promotes the infiltration of effector memory T and CD8+ T cells, as well as the activation of NK cells and the polarization of macrophages to M1. Programmed death 1 blockade antibody combination further enhances the antitumor efficacy of I-Pl FOLactis. In summary, we first develop an innovative intrapleural strategy based on FOLactis, exhibiting remarkable efficacy and favorable biosafety profiles. These findings suggest prospective clinical translation of engineered probiotics for managing MPE through direct administration into the pleural cavity.

A Thermosensitive Bi-Adjuvant Hydrogel Triggers Epitope Spreading to Promote the Anti-Tumor Efficacy of Frameshift Neoantigens.

Tumor-specific frameshift mutations encoding peptides (FSPs) are highly immunogenic neoantigens for personalized cancer immunotherapy, while their clinical efficacy is limited by immunosuppressive tumor microenvironment (TME) and self-tolerance. Here, a thermosensitive hydrogel (FSP-RZ-BPH) delivering dual adjuvants R848 (TLR7/8 agonist) + Zn2+ (cGAS-STING agonist) is designed to promote the efficacy of FSPs on murine forestomach cancer (MFC). After peritumoral injection, FSP-RZ-BPH behaves as pH-responsive sustained drug release at sites near the tumor to effectively transform the immunosuppressive TME into an inflammatory type. FSP-RZ-BPH orchestrates innate and adaptive immunity to activate dendritic cells in tumor-draining lymph nodes and increase the number of FSPs-reactive effector memory T cells (TEM) in tumor by 2.9 folds. More importantly, these TEM also exhibit memory responses to nonvaccinated neoantigens on MFC. This epitope spreading effect contributes to reduce self-tolerance to maintain long-lasting anti-tumor immunity. In MFC suppressive model, FSP-RZ-BPH achieves 84.8% tumor inhibition rate and prolongs the survival of tumor-bearing mice with 57.1% complete response rate. As a preventive tumor vaccine, FSP-RZ-BPH can also significantly delay tumor growth. Overall, the work identifies frameshift MFC neoantigens for the first time and demonstrates the thermosensitive bi-adjuvant hydrogel as an effective strategy to boost bystander anti-tumor responses of frameshift neoantigens.

Micro-to-Nano Oncolytic Microbial System Shifts from Tumor Killing to Tumor Draining Lymph Nodes Remolding for Enhanced Immunotherapy.

Because the tumor-draining lymph nodes (TDLNs) microenvironment is commonly immunosuppressive, oncolytic microbe-induced tumor antigens aren't sufficiently cross-primed tumor specific T cells through antigen-presenting cells (e.g., dendritic cells (DCs)) in TDLNs. Herein, this work develops the micro-to-nano oncolytic microbial therapeutics based on pyranose oxidase (P2 O) overexpressed Escherichia coli (EcP) which are simultaneously encapsulated by PEGylated mannose and low-concentrated photosensitizer nanoparticles (NPs). Following administration, P2 O from this system generates toxic hydrogen peroxide for tumor regression and leads to the release of tumor antigens. The "microscale" EcP is triggered, following exposure to the laser irradiation, to secrete the "nanoscale" bacterial outer membrane vesicles (OMVs). The enhanced TDLNs delivery via OMVs significantly regulates the TDLNs immunomicroenvironment, promoting the maturation of DCs to potentiate tumor antigen-specific T cells immune response. The micro-to-nano oncolytic microbe is leveraged to exert tumor killing and remold TDLNs for initiating potent activation of DCs, providing promising strategies to facilitate microbial cancer vaccination.

Immune characteristics associated with lymph node metastasis in early-stage NSCLC.

Tumor metastasis significantly impacts the prognosis of non-small cell lung cancer (NSCLC) patients,with lymph node (LN) metastasis being the most common and early form of spread. With the development ofadjuvant immunotherapy, increasing attention has been paid to the tumor-draining lymph nodes（TDLN) in early-stageNSCLC, especially tumor-metastatic lymph nodes, which provides poor prognostic information but haspotential benefits in adjuvant treatment. We showed the remodeled immune environment in TDLNs through using TCR-seq to analyse 24 primarylung cancer tissues and 134 LNs from 24 lung cancer patients with or without LN metastasis. Additionally, wecharacterized the spatial profiling of immunocytes and tumor cells in TDLNs and primary tumor sites through usingmulti-IHC. We found the remodeled immune environment in TDLNs through analyzing primary lung cancer tissuesand LNs from NSCLC patients with or without LN metastasis. Considering the intricate communication betweentumor and immunocytes, we further subdivided TDLNs, revealing that metastasis-negative LNs from LN-metastaticpatients (MNLN) exhibited greater immune activation, exhaustion, and memory in comparison to both metastasis-positiveLNs (MPLN) and TDLNs from non-LN-metastatic patients (NMLN). Our data indicate that LN metastasis facilitated tumor-specific antigen presentation in TDLNs andinduces T cell priming, while existing tumor cells generate an immune-suppressive environment in MPLNs throughmultiple mechanisms. These findings contribute to a comprehensive understanding of the immunologicalmechanisms through which LN metastasis influences tumor progression and plays a role in immunotherapy forNSCLC patients.

Cancer cell plasticity and MHC-II-mediated immune tolerance promote breast cancer metastasis to lymph nodes.

Tumor-draining lymph nodes (TDLNs) are important for tumor antigen-specific T cell generation and effective anticancer immune responses. However, TDLNs are often the primary site of metastasis, causing immune suppression and worse outcomes. Through cross-species single-cell RNA-Seq analysis, we identified features defining cancer cell heterogeneity, plasticity, and immune evasion during breast cancer progression and lymph node metastasis (LNM). A subset of cancer cells in the lymph nodes exhibited elevated MHC class II (MHC-II) gene expression in both mice and humans. MHC-II+ cancer cells lacked costimulatory molecule expression, leading to regulatory T cell (Treg) expansion and fewer CD4+ effector T cells in TDLNs. Genetic knockout of MHC-II reduced LNM and Treg expansion, while overexpression of the MHC-II transactivator, Ciita, worsened LNM and caused excessive Treg expansion. These findings demonstrate that cancer cell MHC-II expression promotes metastasis and immune evasion in TDLNs.

Inducible TgfbR1 and Pten deletion in a model of tongue carcinogenesis and chemoprevention.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health problem, with a need for novel approaches to chemoprevention and treatment. Preclinical models that recapitulate molecular alterations that occur in clinical HNSCC patients are needed to better understand molecular and immune mechanisms of HNSCC carcinogenesis, chemoprevention, and efficacy of treatment. We optimized a mouse model of tongue carcinogenesis with discrete quantifiable tumors via conditional deletion of Tgfβr1 and Pten by intralingual injection of tamoxifen. We characterized the localized immune tumor microenvironment, metastasis, systemic immune responses, associated with tongue tumor development. We further determined the efficacy of tongue cancer chemoprevention using dietary administration of black raspberries (BRB). Three Intralingual injections of 500 µg tamoxifen to transgenic K14 Cre, floxed Tgfbr1, Pten (2cKO) knockout mice resulted in tongue tumors with histological and molecular profiles, and lymph node metastasis similar to clinical HNSCC tumors. Bcl2, Bcl-xl, Egfr, Ki-67, and Mmp9, were significantly upregulated in tongue tumors compared to surrounding epithelial tissue. CD4+ and CD8 + T cells in tumor-draining lymph nodes and tumors displayed increased surface CTLA-4 expression, suggestive of impaired T-cell activation and enhanced regulatory T-cell activity. BRB administration resulted in reduced tumor growth, enhanced T-cell infiltration to the tongue tumor microenvironment and robust antitumoral CD8+ cytotoxic T-cell activity characterized by greater granzyme B and perforin expression. Our results demonstrate that intralingual injection of tamoxifen in Tgfβr1/Pten 2cKO mice results in discrete quantifiable tumors suitable for chemoprevention and therapy of experimental HNSCC.

Microbial synthesis of Prussian blue for potentiating checkpoint blockade immunotherapy

Cancer immunotherapy is revolutionizing oncology. The marriage of nanotechnology and immunotherapy offers a great opportunity to amplify antitumor immune response in a safe and effective manner. Here, electrochemically active Shewanella oneidensis MR-1 can be applied to produce FDA-approved Prussian blue nanoparticles on a large-scale. We present a mitochondria-targeting nanoplatform, MiBaMc, which consists of Prussian blue decorated bacteria membrane fragments having further modifications with chlorin e6 and triphenylphosphine. We find that MiBaMc specifically targets mitochondria and induces amplified photo-damages and immunogenic cell death of tumor cells under light irradiation. The released tumor antigens subsequently promote the maturation of dendritic cells in tumor-draining lymph nodes, eliciting T cell-mediated immune response. In two tumor-bearing mouse models using female mice, MiBaMc triggered phototherapy synergizes with anti-PDL1 blocking antibody for enhanced tumor inhibition. Collectively, the present study demonstrates biological precipitation synthetic strategy of targeted nanoparticles holds great potential for the preparation of microbial membrane-based nanoplatforms to boost antitumor immunity.

Harnessing NK cell-based immunotherapy to prevent the high-dose radiotherapy-inducing tumor survival recurrence

Natural killer cells play crucial roles in tumor immunosurveillance and serve as first responders to recognize abnormal cells. Radiotherapy is the mainstay of cancer treatment. However, the effect of high-dose radiotherapy on NK cells remains elusive. Here, we used tumor-bearing mice in the murine colorectal cancer cell line, MC38. The function of NK cells in tumor-draining lymph nodes and tumors was explored after the mice were treated using radiotherapy with 20 Gy and/or blocking antibody αTIGIT at the indicated time. High-dose radiotherapy shaped an immunosuppressive tumor microenvironment to support tumor growth, showing a decreased anti-tumor immunity phenotype in which effector T cells were reduced significantly. Furthermore, the production of functional cytokines and markers in NK cells, including CD107a, granzyme B, and IFN-γ, also remarkably decreased after radiotherapy, while the inhibitory receptor TIGIT was significantly upregulated by FACS analysis. The effect of radiotherapy was significantly elevated after treatment with the combination of radiotherapy and TIGIT inhibition. Moreover, this combination significantly decreased tumor recurrence. Our findings reported that local single high-dose radiotherapy shaped the immunosuppressive microenvironment and inhibited the function of NK cells. Our study revealed compelling evidence suggesting that the enhancement of NK cell function through TIGIT targeting is an effective strategy to mitigate immune suppression caused by high-dose radiotherapy, thereby promoting the inhibition of tumor recurrence.

Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes.

The contribution of different immune cell subsets, especially T cells, in anti-tumor immune response is well established. In contrast to T cells, the anti-tumor contribution of B cells has been scarcely investigated. B-cells are often overlooked, even though they are important players in a fully integrated immune response and constitute a substantial fraction of tumor draining lymph nodes (TDLNs) known also as Sentinel Nodes. In this project, samples including TDLNs, non-TDLNs (nTDLNs) and metastatic lymph nodes from 21 patients with oral squamous cell carcinoma were analyzed by flow cytometry. TDLNs were characterized by a significantly higher proportion of B cells compared with nTDLNs (P = .0127). TDLNs-associated B cells contained high percentages of naïve B cells, in contrary to nTDLNs which contained significantly higher percentages of memory B cells. Patients having metastases in TDLNs showed a significantly higher presence of immunosuppressive B regulatory cells compared with metastasis-free patients (P = .0008). Elevated levels of regulatory B cells in TDLNs were associated with the advancement of the disease. B cells in TDLNs were characterized by significantly higher expression of an immunosuppressive cytokine-IL-10 compared with nTDLNs (P = .0077). Our data indicate that B cells in human TDLNs differ from B cells in nTDLNs and exhibit more naïve and immunosuppressive phenotypes. We identified a high accumulation of regulatory B cells within TDLNs which may be a potential obstacle in achieving response to novel cancer immunotherapies (ICIs) in head and neck cancer.

Galectin-7 reprograms skin carcinogenesis by fostering innate immune evasive programs

Non-melanoma skin cancer (NMSC) has risen dramatically as a result of chronic exposure to sunlight ultraviolet (UV) radiation, climatic changes and clinical conditions associated with immunosuppression. In spite of considerable progress, our understanding of the mechanisms that control NMSC development and their associated molecular and immunological landscapes is still limited. Here we demonstrated a critical role for galectin-7 (Gal-7), a β-galactoside-binding protein preferentially expressed in skin tissue, during NMSC development. Transgenic mice (Tg46) overexpressing Gal-7 in keratinocytes showed higher number of papillomas compared to WT mice or mice lacking Gal-7 (Lgals7−/−) when subjected to a skin carcinogenesis protocol, in which tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were sequentially administered. RNAseq analysis of Tg46 tumor lesions revealed a unique profile compatible with cells of the myelomonocytic lineage infiltrating these tumors, an effect that was substantiated by a higher number of CD11b+Gr1+ cells in tumor-draining lymph nodes. Heightened c-Met activation and Cxcl-1 expression in Tg46 lesions suggested a contribution of this pathway to the recruitment of these cells. Remarkably, Gal-7 bound to the surface of CD11b+Ly6ChiLy6Glo monocytic myeloid cells and enhanced their immunosuppressive activity, as evidenced by increased IL-10 and TGF-β1 secretion, and higher T-cell inhibitory activity. In vivo, carcinogen-treated Lgals7−/− animals adoptively transferred with Gal-7-conditioned monocytic myeloid cells developed higher number of papillomas, whereas depletion of these cells in Tg46-treated mice led to reduction in the number of tumors. Finally, human NMSC biopsies showed increased LGALS7 mRNA and Gal-7 protein expression and displayed transcriptional profiles associated with myeloid programs, accompanied by elevated CXCL1 expression and c-Met activation. Thus, Gal-7 emerges as a critical mediator of skin carcinogenesis and a potential therapeutic target in human NMSC.

CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor

Improvements in tumor immunotherapies depend on better understanding of the anti-tumor Tcell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ Tcells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ Tcells. Murine tumor models revealed that tumor-specific CD8+ Tcells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ Tcell activation in response to cancer is different from that of canonical CD8+ Tcell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ Tcell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.

Regulatory and effector T cell subsets in tumor-draining lymph nodes of patients with squamous cell carcinoma of head and neck

BackgroundA crucial role for the immune system has been proposed in the establishment and progression of head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the cytokine and regulatory profiles of T cells in tumor draining lymph nodes (TDLNs) of patients with HNSCC.ResultsThe frequencies of CD4+TNF-α+ and CD4+TNF-αhi negatively were associated with poor prognostic factors such as LN involvement (P = 0.015 and P = 0.019, respectively), stage of the disease (P = 0.032 and P = 0.010, respectively) and tumor size (P = 0.026 and P = 0.032, respectively). Frequencies of CD8+IFN-γ+ and CD8+IFN-γ+ TNF-α+ T cells showed negative relationship with tumor grade (P = 0.035 and P = 0.043, respectively). While, the frequencies of CD4+IL-4+, CD8+IL-10+, CD8+IL-4+T cells were higher in advanced stages of the disease (P = 0.042, P = 0.041 and P = 0.030, respectively) and CD4+IFN-γ+TNF-α−, CD8+IL-4+ and CD8+IFN-γ+TNF-α− T cells were higher in patients with larger tumor size (P = 0.026 and P = 0.032, respectively). Negative associations were found between the frequencies of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3+CD127low/− Treg cells and cancer stage (P = 0.015 and P = 0.059).ConclusionThis study shed more lights on the changes in immune profile of T cells in TDLNs of HNSCC. Larger tumor size and/or LN involvement were associated with lower frequencies of CD4+TNF-α+, CD8+IFN-γ+ and CD8+IFN-γ+TNF-α+ but higher frequency of CD4+IL-4+ T cells. Moreover, Foxp3+Tregs correlated with good prognostic indicators.

Abscopal Responses to Radiation Combined with Anti-PD-1 and CD122-Directed IL-2/Anti-IL-2 Complexes

<h3>Purpose/Objective(s)</h3> Early clinical trials have provided evidence for RT-induced systemic effects in conjunction with αPD-1 or IL-2 in metastatic patients, but strong abscopal responses are clinically rare. Dual combinations of αPD-1 with more effective and less toxic IL-2 derivatives, e.g., CD122-directed pegylated IL-2, are also currently under investigation. Whether a combination of RT, αPD-1, and CD122-directed IL-2/anti-IL-2 complexes (IL-2c) can increase abscopal effects against established non-irradiated tumors is unknown. Also, in-depth analyses of the differentiation of tumor-specific CD8⁺ T cells have not yet been reported for αPD-1/IL-2c in combination with RT. We investigated how adding IL-2c to hRT/αPD1 affects tumor-specific CD8⁺ T cell differentiation in mouse tumor models and the potential of this triple combination to enhance the abscopal effect compared to the respective dual treatments. <h3>Materials/Methods</h3> Mice bearing bilateral tumors were treated with two fractions of 8 Gy (C51 colon carcinoma model) or 12 Gy (B16 melanoma model); αPD1 was given weekly; IL-2c was given for five consecutive days. CD8 T cell-depleting and CXCR3-blocking antibodies were used to determine if the therapeutic effects depend on CD8⁺ and CXCR3⁺ T cells. Differentiation stages of tumor-specific CD8⁺ T cells in tumor-draining lymph nodes, spleen, blood, and tumors were determined by flow cytometry using MHC-I tetramers and various antibodies. <h3>Results</h3> The abscopal effect was significantly stronger in triple-treated mice compared to mice treated with RT/αPD1 (C51 model: p < 0.01; B16 model: p < 0.05), RT/IL-2c (C51 model: p < 0.01; B16 model: p < 0.001) or αPD1/IL-2c (C51 model: p < 0.0001, B16 model: p < 0.01). Moreover, triple therapy improved survival and resulted in complete cures of 3/12 mice in the C51 model and 2/12 mice in the B16 model. These anti-tumor effects were associated with dramatic expansion of tumor-specific CD8⁺ T cells. Undifferentiated stem-like and effector-like but not terminally differentiated exhausted cells particularly strongly increased. Moreover, IL-2c induced CXCR3 expression on tumor-specific CD8⁺ T cells. Both CD8⁺ (C51 model: p < 0.0001; B16 model: p < 0.01) and CXCR3⁺ (C51 model: p < 0.0001) T cells were crucial for the RT-induced abscopal effect upon RT/αPD-1/IL-2c treatment. <h3>Conclusion</h3> RT/αPD-1/IL-2c triple treatment resulted in superior local and systemic expansion of tumor-specific CD8⁺ T cells with stem- and effector-like phenotypes. Also, IL-2c strongly increased CXCR3⁺ CD8⁺ T cells that were associated with pronounced abscopal responses in models with an established non-irradiated tumor resistant to αPD-1/IL-2c and only transiently responding to RT/αPD-1 or RT/IL-2c. Therefore, such triple combinations appear promising for clinical evaluation in metastatic patients.

Metformin keeps CD8+ T cells active and moving in NASH-HCC immunotherapy

Metformin keeps CD8+ T cells active and moving in NASH-HCC immunotherapy

Abstract 4221: Characterization of OVA-expressing immunogenic syngeneic tumor models for immune checkpoint inhibitor evaluation

Abstract BACKGROUND: Syngeneic mouse tumor models are widely used as preclinical models for immuno-oncology (I/O) research. However, their response to immune checkpoint inhibitors (ICIs) in vivo is limited, despite the increase in tumor infiltrating lymphocytes (TILs), which is potentially due to low intrinsic immunogenicity. Expression of chicken ovalbumin (OVA) engineered into syngeneic tumors has previously been shown to render the cells more immunogenic and increases the response to ICIs such as anti-PD-1. In this study we investigated the potential to increase the response to ICIs through OVA expression and characterized the model for TILs and T cell-mediated immunity compared with parent cell line. METHODS: A series of murine cells were transduced by lentiviral vector (pLVX-EF1a_IRES-PURO) with a chicken OVA coding cDNA. OVA expression was confirmed by western blot following single clone selection under puromycin. MC38-OVA, B16-F10-OVA, CT26.WT-OVA and EMT6-OVA cells were subcutaneously inoculated into immunocompetent C57BL/6 or BALB/c mice to evaluate the tumorigenicity and their in vivo response to anti-PD-1(10mg/kg, IP) treatment. In the study, blood, spleen and tumors were collected at termination for FACS analysis of the immune cell populations and the OVA-specific T cells. The effects of CD4+ and CD8+ T lymphocyte depletion was investigated using anti-CD4 and anti-CD8 antibodies in the PD-1 cured EMT6-OVA mice. Adoptive cell transfer (ACT) was evaluated by re-challenge studies in both OVA expressing and WT tumor-bearing mice with isolated T cells derived from OVA untreated mice, anti-PD-1 cured mice or OT-I mice. RESULTS: Anti-PD-1 treatment resulted in complete regression in most mice bearing CT26.WT-OVA, MC38-OVA and EMT6-OVA, whereas an improvement in the response was shown in B16-F10-OVA in vivo. T cell and NK cells expansion was observed after anti-PD-1 treatment in peripheral blood with increased T cells, Treg cells and NK cells in both tumor-draining lymph nodes and spleen. In the re-challenge study anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited OVA expressing tumor growth following ACT. CD3+ T cells from EMT6-OVA-bearing mice showed anti-tumor immunity in vivo. CD4 and CD8 depletion studies provided further insight into the T cell role in PD-1 treatment. CONCLUSIONS: The panel of OVA expressing murine cell lines displayed increased T cell-mediated immunity and enhanced anti-PD-1 responses compared to WT models for different cancer indications in vivo, providing novel immunogenic syngeneic models for preclinical immunotherapy evaluation. Citation Format: Chenpan Nie, Yi Zhang, Jia Zheng, Annie X. An, Jun Zhou, Henry Q.X. Li, Jingjing Wang. Characterization of OVA-expressing immunogenic syngeneic tumor models for immune checkpoint inhibitor evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4221.

Innate lymphoid cells: NK and cytotoxic ILC3 subsets infiltrate metastatic breast cancer lymph nodes

ABSTRACT Innate lymphoid cells (ILCs) – which include cytotoxic Natural Killer (NK) cells and helper-type ILC – are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127− CD56− NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality.

In Situ Tumor Vaccination with Calcium-Linked Degradable Coacervate Nanocomplex Co-Delivering Photosensitizer and TLR7/8 Agonist to Trigger Effective Anti-Tumor Immune Responses.

In situ anti-tumor vaccination is an attractive type of cancer immunotherapy which relies on the effectiveness of dendritic cells (DCs) to engulf tumor antigens, become activated, and present antigens to T cells in lymphoid tissue. Here, a multifunctional nanocomplex based on calcium crosslinked polyaspartic acid conjugated to either a toll-like receptor (TLR)7/8 agonist or a photosensitizer is reported. Intratumoral administration of the nanocomplex followed by laser irradiation induces cell killing and hence generation of a pool of tumor-associated antigens, with concomitant promotion of DCs maturation and expansion of T cells in tumor-draining lymph nodes. Suppression of tumor growth is observed both at the primary site and at the distal site, thereby hinting at successful induction of an adaptive anti-tumor response. This strategy holds promise for therapeutic application in a pre-operative and post-operative setting to leverage to mutanome of the patient's own tumor to mount immunological memory to clear residual tumor cells and metastasis.

Correlation and prognostic implications of intratumor and tumor draining lymph node Foxp3+ T regulatory cells in colorectal cancer

BackgroundThe prognostic value of intratumor T regulatory cells (Tregs) in colorectal cancer (CRC) was previously reported, but the role of these cells in tumor draining lymph nodes (TDLNs) was less addressed.MethodsA total of 150 CRC stages I-IV were retrospectively enrolled. Intratumor and TDLN Tregs were examined by immunohistochemical assay. The association of these cells was estimated by Pearson correlation. Survival analyses of subgroups were conducted by Kaplan–Meier curves, and the log-rank test and risk factors for survival were tested by the Cox proportional hazard model.ResultsHigh accumulation of Tregs in tumors was significant in patients with younger age and good histological grade, where enrichment of these cells in TDLNs was more apparent in those with node-negative disease and early TNM stage disease, both of which were more common in early T stage cases. A significant correlation of intratumoral and TDLN Tregs was detected. Patients with higher intratumoral Tregs displayed significantly better PFS and OS than those with lower Tregs. However, no such differences were found, but a similar prognostic prediction trend was found for these cells in TDLNs. Finally, intratumoral Tregs were an independent prognostic factor for both PFS (HR = 0.97, 95% CI 0.95–0.99, P < 0.01) and OS (HR = 0.98, 95% CI 0.95–1.00, P = 0.04) in the patients.ConclusionsHigher intratumor Tregs were associated with better survival in CRC. Although no such role was found for these cells in TDLNs, the positive correlation and similar prognostic prediction trend with their intratumoral counterparts may indicate a parallelized function of these cells in CRC.

Regorafenib enhances antitumor immune efficacy of anti-PD-L1 immunotherapy on oral squamous cell carcinoma

Oral squamous cells carcinoma (OSCC) is the most common oral malignancy that majorly originated from oral cavity. Though the prognostic and predictive value of targeting checkpoint molecules has been reported on OSCC, the treatment efficacy of monotherapy is remaining limited. Several studies suggested that multikinase inhibitors may show potential to facilitate anti-PD-L1-induced anti-tumor immunity. Regorafenib, an oral multikinase inhibitor has been approved by FDA for various types of cancer treatment. Here, we aim to identify whether regorafenib may boost anti-tumor immunity of anti-PD-L1 in MOC1-bearing OSCC animal model. The alteration of immune cells such as M1/M2-like macrophages (MΦ), cytotoxicity T cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) after combination of anti-PD-L1 and regorafenib was validated by flow cytometry and immunohistochemistry staining. The combination index analysis (CI=0.89) supported that regorafenib effectively induce anti-OSCC efficacy of anti-PD-L1. Combination of anti-PD-L1 and regorafenib may not only trigger the polarization of M1-like MΦ (CD11b+CD86+) in mice bone marrow (BM) and spleen (SP), but also induce the accumulation and function of CD8+ T cells in tumor-draining lymph node (TDLN) and tumor. In addition, immunosuppressive related cells (MDSCs and Treg) and factors were all decreased by combination therapy in BM, SP and tumor. In sum, regorafenib may improve anti-OSCC efficacy of anti-PD-L1 through systemically and locally upregulating the immunostimulation immunity and suppressing immunosuppression immunity.