Activation Of Anti-tumor Immune Response Research Articles

Abstract PO2-03-11: A novel peripheral CD4+ T cell immune network associated with response to PD-1 inhibitor in early-stage triple negative breast cancer

Abstract A novel peripheral CD4+ T cell immune network associated with response to PD-1 inhibitor in early-stage triple negative breast cancer Background: Immunotherapy has showed benefits in early-stage triple-negative breast cancer (eTNBC). However, there is an urgent need to identify novel biomarkers that can guide stratification for immunotherapy in eTNBC. Anti-tumor immunity is a systemic response that encompasses the mobilization of dendritic cells, lymphocytes, and myeloid cells involving the tumor, blood, and lymph nodes. The activation of anti-tumor immune responses via immunotherapy is expected to reflect in the subset change of circulation. Combined with advantage of dynamic monitoring by liquid biopsy, we detected peripheral blood in the eTNBC patients under immunotherapy to explore potential immune therapeutic predictive subsets. Methods: Single-cell (sc) RNA sequencing was utilized to determine the transcriptomics of immune cells in the peripheral blood. Meanwhile scTCR sequencing was used to trace the dynamic changes of clonetypes under multi-therapy. Nanostring analysis of primary tumor and in vivo models were used to validate the mechanism of immune crosstalk. Prediction index was built based on the proportion of immune subsets at each time point and change of tumor size in each patient. Results: 12 eTNBC patients who participated in neoadjuvant chemotherapy in combination with PD-1 inhibitor (Toripalimab) clinical trial were included in this study. The clinical trial consists of two stages. All eligible patients received four cycles of EC (cyclophosphamide plus epirubicin) regimen and followed four cycles of nab-paclitaxel plus PD-1 inhibitor. Among the 12 patients, 5 patients achieved pCR, while the remaining 7 patients were non-pCR. Peripheral blood samples were collected at baseline, after chemotherapy induction, and on PD-1 inhibitor treatment for scRNA- and scTCR-sequencing. Through prediction index, a novel predictive pattern based on ratio of memory T cells (central memory T and resident memory T) to effective T cells in the peripheral was vividly defined, by which the patients could be classified into C-type (memory T cells dominated) and E-type (effective T cells dominated). The C-type indicated better efficacy of immunotherapy combination therapy, while the E-type suggested better efficacy of chemotherapy alone. Meanwhile, a novel peripheral CD4+Trm (resident-memory-like) subset that possessed both strong differentiation potential and memory function was higher in pCR group at baseline. TCR-tracing analysis revealed that the new clones induced by chemotherapy mainly belong to the CD4+ Trm and CD8+ CD161+ Tcm (central memory) subsets, and the upregulation of CD8+ CD161+ Tcm during therapy was a dynamic predictor for immunotherapy benefits. Besides, most of CD4+Trm transitioned into CD4+Th1-like cells as an activated status, which considered CD4+Trm as a group of reservoirs with polyclonality and differentiation potential for tumor-killing. Meanwhile, the increase in CD5+ cDC2 (type 2 conventional dendritic cell) after chemotherapy was associated with enhanced antigen-presenting function and cell communication and co-stimulation with CD4+ Trm cells which helped the activated transition from CD4+Trm into CD4+Th1-like cells in lymph nodes. Moreover, CD4+Th1-like cells increased secretion of Th1 cytokines thus leading to promote CD8+CD161+ Tcm upregulation and tumor-infiltration after immunotherapy. Conclusions: A novel peripheral pattern based on subsets of T cells as C-type and E-type was established as potential predictors for immunotherapy stratification in eTNBC patients. Meanwhile, a novel peripheral CD4+Trm immune network was identified, in which CD5+cDC2 activated the transition of CD4+Trm into CD4+Th1-like subset leading to activation and tumor-infiltration of CD8+CD161+Tcm. All the above provided potential therapeutic targets for enhancing immunotherapy. Citation Format: Zehao Wang, Zhibo Shao, Bingqiu Xiu, Jiong Wu. A novel peripheral CD4+ T cell immune network associated with response to PD-1 inhibitor in early-stage triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-03-11.

Fasting and fasting-mimicking conditions in the cancer immunotherapy era.

Fasting and fasting-mimicking conditions modulate tumor metabolism and remodel the tumor microenvironment (TME), which could be exploited for the treatment of tumors. A body of evidence demonstrates that fasting and fasting-mimicking conditions can kill cancer cells, or sensitize them to the antitumor activity of standard-of-care drugs while protecting normal cells against their toxic side effects. Pre- and clinical data also suggest that immune responses are involved in these therapeutic effects. Therefore, there is increasing interest in evaluating the impact of fasting-like conditions in the efficacy of antitumor therapies based on the restoration or activation of antitumor immune responses. Here, we review the recent progress in the intersection of fasting-like conditions and current cancer treatments, with an emphasis on cancer immunotherapy.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

BackgroundOvarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient...

Heterogeneous expression of ARID1A in colorectal cancer indicates distinguish immune landscape and efficacy of immunotherapy

ObjectiveAT-rich interaction domain 1A (ARID1A) mutant tumors show active anti-tumor immune response, which is the potential indication of immunotherapy. However, the relationship between the heterogeneous ARID1A expression and the immune response and immunotherapy efficacy in colorectal cancer (CRC) is still unclear.MethodsWe collected 1113 cases of patients with stage I-IV CRC who underwent primary resection at Harbin Medical University Cancer Hospital. ARID1A expression in CRC tissues was assessed via immunohistochemistry (IHC). CD8, CD163 and FOXP3 were stained by IHC to identify the immune landscape. Clinicopathological features of patients were compared using statistical tests like the Wilcoxon-Mann–Whitney test or χ2 tests. Kaplan–Meier survival analysis with log-rank tests were employed.ResultsHeterogeneous ARID1A expression was categorized into integrity expression, complete expression deficiency (cd-ARID1A), partial expression deficiency (pd-ARID1A), and clonal expression deficiency (cld-ARID1A). ARID1A-deficient expression was significant association with dMMR (P value < 0.001). Patients with ARID1A deficiency, compared to ARID1A-proficient patients, exhibited increased infiltration levels of CD8 + P value < 0.0001), CD163 + P value < 0.001), and FOXP3 + P value < 0.001).cells within the tumor tissue. However, in different subgroups, only samples with complete or partial deficiency of ARID1A showed a higher abundance of lymphocyte infiltration. In patients with ARID1A-clonal expression deficiency tumor, the infiltration patterns of three immune cell types were comparable to those in ARID1A-proficient patients. Heterogeneous ARID1A expression is related to the different prognosis and immunotherapythe efficacy in CRC patients.ConclusionHeterogeneous ARID1A expression is accompanied by a different immune landscape. CRC patients with ARID1A-clonal expression deficiency do not benefit from the treatment of immune checkpoint inhibitors (ICIs).

Promises of Protein Kinase Inhibitors in Recalcitrant Small-Cell Lung Cancer: Recent Scenario and Future Possibilities.

SCLC is refractory to conventional therapies; targeted therapies and immunological checkpoint inhibitor (ICI) molecules have prolonged survival only marginally. In addition, ICIs help only a subgroup of SCLC patients. Different types of kinases play pivotal roles in therapeutics-driven cellular functions. Therefore, there is a significant need to understand the roles of kinases in regulating therapeutic responses, acknowledge the existing knowledge gaps, and discuss future directions for improved therapeutics for recalcitrant SCLC. Here, we extensively review the effect of dysregulated kinases in SCLC. We further discuss the pharmacological inhibitors of kinases used in targeted therapies for recalcitrant SCLC. We also describe the role of kinases in the ICI-mediated activation of antitumor immune responses. Finally, we summarize the clinical trials evaluating the potential of kinase inhibitors and ICIs. This review overviews dysregulated kinases in SCLC and summarizes their potential as targeted therapeutic agents. We also discuss their clinical efficacy in enhancing anticancer responses mediated by ICIs.

Activation of RIG-I/MDA5 Signaling and Inhibition of CD47-SIRPα Checkpoint with a Dual siRNA-Assembled Nanoadjuvant for Robust Cancer Immunotherapy.

Antigen-presenting cells (APCs) play a crucial role in the anti-tumor immunity as they are responsible for capturing, processing, and presenting tumor antigens to T cells. However, their activation is often limited by the absence of adjuvants and the suppressive effects of immune checkpoints, such as CD47-SIRPα. Herein, we present a nanoadjuvant that is self-assembled from long RNA building blocks generated through rolling circle transcription (RCT) reaction and further modified with cationic liposomes. Owing to the high load of densely packed RNA, this nanoadjuvant could robustly activate RIG-I/MDA5 signaling in APCs, leading to the maturation of dendritic cells (DCs) and the polarization of tumor-associated macrophages (TAMs) toward an anti-tumor M1-like phenotype. In addition, with a well-designed template, the generated long RNA from RCT reaction includes two kinds of siRNA targeting both CD47 in tumor cells and SIRPα in APCs. This dual gene silencing results in efficient inhibition of the CD47-SIRPα checkpoint. Collectively, the robust activation of RIG-I/MDA5 signaling and efficient inhibition of CD47-SIRPα checkpoint enhance the phagocytic activity of APCs, which in turn promotes the cross-priming of effector T cells and the activation of anti-tumor immune responses. This study therefore provides a simple and robust RNA nanoadjuvant for cancer immunotherapy.

Identification and characterization of immunogenic neoantigens in colorectal cancer (CRC).

161 Background: Presentation of neoantigens, short peptides derived from tumor-specific somatic mutations, by HLA molecules triggers the activation of anti-tumor immune response. Recognition of neoantigens by autologous T cells promotes sensitivity to immune checkpoint inhibitors (ICI) in mismatch repair deficient (MMRd)/microsatellite instability high (MSI-H) tumors. Neoantigen-targeted reactivity has also been reported in microsatellite stable (MSS) tumors, and neoantigens are emerging targets for tumor vaccines and adoptive cell therapy. Here, we aimed to comprehensively assess the spectrum of immunogenic neoantigens in CRC to identify new subpopulations for these therapeutic strategies. Methods: 7,053 CRC tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (720-gene panel) and RNA (whole transcriptome) were analyzed. MSI status was determined by immunohistochemistry of MMR proteins and/or NGS. Immune epitope prediction was performed on translated peptide sequences harboring detected mutations using the NetMHCpan v4.0 method in the Immune Epitope Database, with HLA genotyping performed using arcasHLA. Immune/stromal cell abundance in the tumor microenvironment (TME) was estimated from RNA expression profiles using MCP-Counter. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed signature, TIS). Results: 6,600,005 unique peptide:allele interactions were tested, of which 368,468 (5.6%) were predicted neoantigens with patient-specific HLA alleles, including 16,184 (0.25%) with high affinity. MMRd/MSI-H tumors (6.9%) had higher neoantigen load at all affinity levels compared to MSS, with 10,243 (63.3%) and 5,621 (34.7%) of the predicted high affinity neoantigens exclusively associated with MSS and MMRd/MSI-H, respectively. 32 recurrent high affinity neoantigens were identified in &gt; 10 samples, most frequently derived from APC mutations in MSS tumors (78% of recurrent neoantigens), and from RNF43 (25%), ASXL1 (20%), and KMT2D (15%) in MMRd/MSI-H. TIS scores positively correlated with the abundance of various immune cell populations in the TME, notably cytotoxic lymphocytes (r = 0.46). Among the recurrent high affinity neoantigens, those with the highest mean TIS scores (&gt; 90th percentile) resulted from mutation of MSH3 (K383fs, 5.1%) and KMT2D (multiple variants, 2.8%) in MMRd/MSI-H CRC, while those in MSS CRC derived from SOX9 (multiple variants, 0.2%) and APC (multiple variants, 0.7%) but were not associated with increased mean TIS scores (~55th percentile). Conclusions: This is one of the largest studies to investigate the landscape of immunogenic neoantigens in CRC. We were able to identify candidate recurrent peptides with high HLA binding affinity and an association with a positive TIS signature supporting the role of neoantigens as potential cancer immunotherapy targets.

Phospholipid scramblase 1 is involved in immunogenic cell death and contributes to dendritic cell–based vaccine efficiency to elicit antitumor immune response in vitro

Background aimsWhole tumor cell lysates (TCLs) obtained from cancer cells previously killed by treatments able to promote immunogenic cell death (ICD) can be efficiently used as a source of tumor-associated antigens for the development of highly efficient dendritic cell (DC)-based vaccines. Herein, the potential role of the interferon (IFN)-inducible protein phospholipid scramblase 1 (PLSCR1) in influencing immunogenic features of dying cancer cells and in enhancing DC-based vaccine efficiency was investigated. MethodsPLSCR1 expression was evaluated in different mantle-cell lymphoma (MCL) cell lines following ICD induction by 9-cis-retinoic acid (RA)/IFN-α combination, and commercial kinase inhibitor was used to identify the signaling pathway involved in its upregulation. A Mino cell line ectopically expressing PLSCR1 was generated to investigate the potential involvement of this protein in modulating ICD features. Whole TCLs obtained from Mino overexpressing PLSCR1 were used for DC loading, and loaded DCs were employed for generation of tumor antigen-specific cytotoxic T lymphocytes. ResultsThe ICD inducer RA/IFN-α combination promoted PLSCR1 expression through STAT1 activation. PLSCR1 upregulation favored pro-apoptotic effects of RA/IFN-α treatment and enhanced the exposure of calreticulin on cell surface. Moreover, DCs loaded with TCLs obtained from Mino ectopically expressing PLSCR1 elicited in vitro greater T-cell–mediated antitumor responses compared with DCs loaded with TCLs derived from Mino infected with empty vector or the parental cell line. Conversely, PLSCR1 knock-down inhibited the stimulating activity of DCs loaded with RA/IFN-α–treated TCLs to elicit cyclin D1 peptide-specific cytotoxic T lymphocytes. ConclusionsOur results indicate that PLSCR1 improved ICD-associated calreticulin exposure induced by RA/IFN-α and was clearly involved in DC-based vaccine efficiency as well, suggesting a potential contribution in the control of pathways associated to DC activation, possibly including those involved in antigen uptake and concomitant antitumor immune response activation.

Modulation of SLFN11 induces changes in DNA Damage response in breast cancer

BackgroundLack of Schlafen family member 11 (SLFN11) expression has been recently identified as a dominant genomic determinant of response to DNA damaging agents in numerous cancer types. Thus, several strategies aimed at increasing SLFN11 are explored to restore chemosensitivity of refractory cancers. In this study, we examined various approaches to elevate SLFN11 expression in breast cancer cellular models and confirmed a corresponding increase in chemosensitivity with using the most successful efficient one. As oncogenic transcriptomic downregulation is often driven by methylation of the promotor region, we explore the demethylation effect of 5-aza-2′-deoxycytidine (decitabine), on the SLFN11 gene. Since SLFN11 has been reported as an interferon inducible gene, and interferon is secreted during an active anti-tumor immune response, we investigated the in vitro effect of IFN-γ on SLFN11 expression in breast cancer cell lines. As a secondary approach to pick up cross talk between immune cells and SLFN11 expression we used indirect co-culture of breast cancer cells with activated PBMCs and evaluated if this can drive SLFN11 upregulation. Finally, as a definitive and specific way to modulate SLFN11 expression we implemented SLFN11 dCas9 (dead CRISPR associated protein 9) systems to specifically increase or decrease SLFN11 expression.ResultsAfter confirming the previously reported correlation between methylation of SLFN11 promoter and its expression across multiple cell lines, we showed in-vitro that decitabine and IFN-γ could increase moderately the expression of SLFN11 in both BT-549 and T47D cell lines. The use of a CRISPR-dCas9 UNISAM and KRAB system could increase or decrease SLFN11 expression significantly (up to fivefold), stably and specifically in BT-549 and T47D cancer cell lines. We then used the modified cell lines to quantify the alteration in chemo sensitivity of those cells to treatment with DNA Damaging Agents (DDAs) such as Cisplatin and Epirubicin or DNA Damage Response (DDRs) drugs like Olaparib. RNAseq was used to elucidate the mechanisms of action affected by the alteration in SLFN11 expression. In cell lines with robust SLFN11 promoter methylation such as MDA-MB-231, no SLFN11 expression could be induced by any approach.ConclusionTo our knowledge this is the first report of the stable non-lethal increase of SLFN11 expression in a cancer cell line. Our results show that induction of SLFN11 expression can enhance DDA and DDR sensitivity in breast cancer cells and dCas9 systems may represent a novel approach to increase SLFN11 and achieve higher sensitivity to chemotherapeutic agents, improving outcome or decreasing required drug concentrations. SLFN11-targeting therapies might be explored pre-clinically to develop personalized approaches.

Potential biomarkers for predicting immune response and outcomes in lung cancer patients undergoing thermal ablation.

Thermal ablation is a promising alternative treatment for lung cancer. It disintegrates cancer cells and releases antigens, followed by the remodeling of local tumor immune microenvironment and the activation of anti-tumor immune responses, enhancing the overall effectiveness of the treatment. Biomarkers can offer insights into the patient's immune response and outcomes, such as local tumor control, recurrence, overall survival, and progression-free survival. Identifying and validating such biomarkers can significantly impact clinical decision-making, leading to personalized treatment strategies and improved patient outcomes. This review provides a comprehensive overview of the current state of research on potential biomarkers for predicting immune response and outcomes in lung cancer patients undergoing thermal ablation, including their potential role in lung cancer management, and the challenges and future directions.

Tumor microenvironment-responsive delivery nanosystems reverse immunosuppression for enhanced CO gas/immunotherapy.

Carbon monoxide (CO) gas therapy demonstrates great potential to induce cancer cell apoptosis and antitumor immune responses, which exhibits tremendous potential in cancer treatment. However, the therapeutic efficacy of CO therapy is inhibited by the immunosuppressive tumor microenvironment (TME). Herein, a facile strategy is proposed to construct hollow-structured rough nanoplatforms to boost antitumor immunity and simultaneously reverse immunosuppression by exploring intrinsic immunomodulatory properties and morphological optimization of nanomaterials. The TME-responsive delivery nanosystems (M-RMH) are developed by encapsulating the CO prodrug within hollow rough MnO2 nanoparticles and the subsequent surface functionalization with hyaluronic acid (HA). Rough surfaces are designed to facilitate the intrinsic properties of HA-functionalized MnO2 nanoparticles (RMH) to induce dendritic cell maturation and M1 macrophage polarization by STING pathway activation and hypoxia alleviation through enhanced cellular uptake. After TME-responsive degradation of RMH, controlled release of CO is triggered at the tumor site for CO therapy to activate antitumor immunity. More importantly, RMH could modulate immunosuppressive TME by hypoxia alleviation. After the combination with aPD-L1-mediated checkpoint blockade therapy, robust antitumor immune responses are found to inhibit both primary and distant tumors. This work provides a facile strategy to construct superior delivery nanosystems for enhanced CO/immunotherapy through efficient activation of antitumor immune responses and reversal of immunosuppression.

Recent Advances in Molecular Mechanisms of Cancer Immunotherapy

Cancer is among the current leading causes of death worldwide, despite the novel advances that have been made toward its treatment, it is still considered a major public health concern. Considering both the serious impact of cancer on public health and the significant side effects and complications of conventional therapeutic options, the current strategies towards targeted cancer therapy must be enhanced to avoid undesired toxicity. Cancer immunotherapy has become preferable among researchers in recent years compared to conventional therapeutic options, such as chemotherapy, surgery, and radiotherapy. The understanding of how to control immune checkpoints, develop therapeutic cancer vaccines, genetically modify immune cells as well as enhance the activation of antitumor immune response led to the development of novel cancer treatments. In this review, we address recent advances in cancer immunotherapy molecular mechanisms. Different immunotherapeutic approaches are critically discussed, focusing on the challenges, potential risks, and prospects involving their use.

Effect of epidural blockade on immune parameters in the perioperative period: randomized controlled study

BACKGROUND: The potential benefits of anaesthesia techniques in the parameters of cellular immunity and prevention of metastasis in various types of cancer are attracting increasing attention.&#x0D; OBJECTIVE: Our aim was to estimate the influence of the epidural blockade as a component of the anesthesia support on the immunity indexes in the patients operated for gastric cancer.&#x0D; MATERIALS AND METHODS: 89 patients operated on for gastric cancer, divided into two groups were investigated: in the 1st group (n=27) there was general anesthesia (GA) in combination with extended epidural blockade; in the 2nd group (n=24) GA with postoperative intravenous administration of analgesics. T-cell counts (CD3+, CD4+ CD8+, CD4+/CD8+) and cytokines (IL-4, IL-6, IL-10, INF-y and TNF-a) were determined.&#x0D; RESULTS: The CD3+ T-cell levels in both groups were significantly (р 0.05) reduced immediately after surgery. On the 2nd and 3rd day the decrease in CD3+ T-cells was more pronounced (р 0.05) in the 2nd group compared to the 1st group. The content of CD4+ T-cells decreased by the 1st day of the postoperative period (р 0,05) but then gradually increased, the CD4+ level in the 1st group was significantly (р 0.05) higher than in the 2nd group. The changes of CD8+ content between the studied groups were not statistically significant. The immunoregulatory index in both groups decreased by1st day (р 0.05). On the 9th day the index in the 1st group returned to the basic values, while in the 2nd group it remained significantly lower. On the 1st and 3rd day, the levels of IL-4 and IL-6 significantly (р 0.05) increased, while in the 2nd group the levels were higher than in the 1st group (р 0.05). On the 9th day IL-4 and IL-6 indices returned to their initial values. The changes of IL-10 level had a similar pattern, while in the 1st group the rate was significantly higher (р 0.05). At all stages of investigation, the level of IFN- was significantly (р 0.05) higher in the 2nd group, the level of TNF- was higher in the 1st group (р 0.05).&#x0D; CONCLUSION: The use of postoperative epidural analgesia during gastric resection for malignant neoplasm is associated with a less suppressive effect on the content of subpopulations of T-lymphocytes and cytokines in comparison with intravenous administration of analgesics and may contribute to the activation of antitumor immune response. Prospective long-term studies are needed to evaluate physician epidural analgesia for oncological outcomes after surgery for gastric cancer.

Abstract 6408: Anti-tumor immunity evolved by novel titanium peroxide nanoparticles (TiOxNPs) as a radiosensitizer and PD-1 blockade

Abstract Abstract: Release and presentation of tumor antigens are the preconditions of cellular immune response. Radiotherapy have been reported to release endogenous tumor antigens and enhanced the adjuvanticity from dying cancer cells via endoplasmic reticulum (ER) stress, known as immunogenic cell death. We developed a novel radiotherapy using radiosensitizer (TiOxNPs) that could drive effective anti-tumor responses. The TiOxNPs were robustly radiation-induced reactive oxygen species (ROS) generation. This burst therapy could enhance to release and presentation of endogenous tumor antigens, subsequently cross-presentation of antigens by Dendritic cells (DCs) for T-cell activation in secondary lymphoid organs (SLO). Methods: B16-F10 murine melanoma cells (5 × 105) were subcutaneously injected into the right flank of C57BL/6J respectively. Mice were divided into five treatment groups: 1) untreated; 2) XRT; 3) XRT+ TiOxNPs; 4) XRT+anti-PD-1Ab; and 5) XRT+ TiOxNPs +anti-PD-1Ab. B16-F10 tumors were intratumorally injected with TiOxNPs (8mg/kg) on days 10 and received 5Gy or 10Gy X-ray radiation approximately 1 hour after the injection. Anti-PD-1 antibody (200µg) intraperitoneally administered to the mice on the first day of X-ray irradiation, days 13 and 16. The tumor volume were assessed every 2-3 days after treatment. Mice were sacrificed after days 17 and isolated spleen and tumor. Results: TiOxNPs acted as a potent radiosensitizer, which enhanced cytotoxic ROS-mediated cell death. This combination effect could not only a local treatment that directly kills cancer cells but also could enhance the adjuvanticity from dying cancer cells by DAMPs, and significantly upregulate expression of MHC class I on tumor cells. Treatment of B16-F10 melanoma tumor model with burst therapy resulted in tumor shrinkage and extended overall survival. Burst therapy facilitated the recruitment and maturation of DCs into SLO, and improved antigen presentation to naive T cells. Infiltration into the tumor site of CTL were correlated with increasing of migration DCs into SLO. Conclusion: A novel radiotherapy combining with TiOxNPs played an important role in release of endogenous tumor antigens and DAMPs. The upregulation of MHC class I expression on tumor cells after X-ray irradiation facilitated the recognition of the tumor cells by T cells, leading to significantly enhanced antitumor immune response compared to exisiting immunoradiotherapy. This novel nanoparticles exposed to X-ray irradiation could trigger of antigen-specific immune responses and transform a tumor into a site for activation of antitumor immune response. Citation Format: Yoshiko Fujita, Hikaru Kubota, Chiaki Ogino, Ryohei Sasaki. Anti-tumor immunity evolved by novel titanium peroxide nanoparticles (TiOxNPs) as a radiosensitizer and PD-1 blockade [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 6408.

Abstract 4614: Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest tumors in humans. PDAC has an immunosuppressive tumor microenvironment, constituting for poor immunotherapy outcomes. Lymphatic vessels (LVs) transport leukocytes and antigens from tumor peripheries to draining lymph nodes (dLNs) for activation of anti-tumor immune responses. It is believed that impairment of the lymphatic function may result in delayed and ineffective immune activation against tumors. In this study, we created a three-dimensional (3D) tumor-on-chip model to examine phenotypical and functional changes in 3D engineered LVs, composed of human primary lymphatic endothelial cells (LECs) in co-culture with PDAC cells. Our in vitro studies revealed that PDAC cells induce lymphangiogenesis and tighten cell-cell junctions in LECs, forming “zipper-like” LEC junctions, compared to control LECs with “button-like” junctions. We introduced 70kDa dextran into the LV lumens and showed PDAC-induced zippering of junctions decreased vessel permeability. These results led us to hypothesize that altered LEC junctions may affect fluid drainage into the LVs in vivo. We inoculated PDAC cells into the mouse ear pinnae and performed whole-mount immunostaining and lymphatic drainage experiment by injecting Evans blue into the ears and measured the remained Evans blue in the ears. In the ear experiments, inoculated PDAC cells affected host LVs with originally button-like junctions to become zipper-like and impede lymphatic drainage. To identify a potential signaling pathway that may be responsible for LEC junction zippering, we used phospho-kinase arrays and discovered that PDAC cells induced LECs to increase phosphorylation of GSK3-β, JNK kinases, and enhanced the total protein level of β-catenin, implying that PDAC cells turned on the WNT signaling in LECs. In preliminary screening, we treated LECs co-cultured with PDAC cells with WNT-C59, an WNT inhibitor that blocks secretion of all WNT ligands, which resulted in decreased number of LV sprouting and less contractile actin-cytoskeleton in LECs. It is critical to know which molecules were secreted by PDAC cells and influenced the WNT signaling in LECs. With that in mind, we plan to perform mass spectrometry with PDAC-conditioned media to identify PDAC-specific secretome; and run RNA-seq with LECs isolated from mouse ears with or without PDAC to investigate molecular basis of the WNT activation in LECs and LV remodeling. Finally, we plan to examine the egress of leukocytes from primary PDAC tumor site, T cell activation in tumor-dLNs, and PDAC tumor growth with or without WNT inhibition in Kaeda mice. The identity and function of PDAC-egressed immune cells, and those which stayed within the primary tumor will enhance our understanding of the roles of LVs in orchestrating anti-tumor immunity in PDAC. Citation Format: Anna Maria Kolarzyk, Conor Loy, Renhao Lu, Iwijn De Vlaminck, Deborah Fowell, Esak Lee. Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models. [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 4614.

Magnetic-Acoustic Sequentially Actuated CAR T Cell Microrobots for Precision Navigation and In Situ Antitumor Immunoactivation.

Despite its clinical success, chimeric antigen receptor T (CAR T)-cell immunotherapy remains limited in solid tumors, owing to the harsh physical barriers and immunosuppressive microenvironment. Here a CAR-T-cell-based live microrobot (M-CAR T) is created by decorating CAR T with immunomagnetic beads using click conjugation. M-CAR Ts are capable of magnetic-acoustic actuation for precision targeting and in situ activation of antitumor immune responses. Sequential actuation endows M-CAR Ts with magnetically actuated anti-flow and obstacle avoidance as well as tissue penetration driven by acoustic propulsion, enabling efficient migration and accumulation in artificial tumor models. In vivo, sequentially actuated M-CAR Ts achieves long-distance targeting and accumulate at the peritumoural area under programmable magnetic guidance, and subsequently acoustic tweezers actuate M-CAR Ts to migrateinto deep tumor tissues, resulting in a 6.6-fold increase in accumulated exogenous CD8+ CAR T cells compared with that without actuation. Anti-CD3/CD28 immunomagnetic beads stimulate infiltrated CAR T proliferation and activation in situ, significantly enhancing their antitumor efficacy. Thus, thissequential-actuation-guided cell microrobot combines the merits of autonomous targeting and penetration of intelligent robots with in situ T-cell immunoactivation, and holds considerable promise for precision navigation and cancer immunotherapies.

Dual-Cascade Activatable Nanopotentiators Reshaping Adenosine Metabolism for Sono-Chemodynamic-Immunotherapy of Deep Tumors.

Immunotherapy is an attractive treatment strategy for cancer, while its efficiency and safety need to be improved. A dual-cascade activatable nanopotentiator for sonodynamic therapy (SDT) and chemodynamic therapy (CDT)-cooperated immunotherapy of deep tumors via reshaping adenosine metabolism is herein reported. This nanopotentiator (NPMCA ) is constructed through crosslinking adenosine deaminase (ADA) with chlorin e6 (Ce6)-conjugated manganese dioxide (MnO2 ) nanoparticles via a reactive oxygen species (ROS)-cleavable linker. In the tumor microenvironment with ultrasound (US) irradiation, NPMCA mediates CDT and SDT concurrently in deep tumors covered with 2-cm tissues to produce abundant ROS, which results in dual-cascade scissoring of ROS-cleavable linkers to activate ADA within NCMCA to block adenosine metabolism. Moreover, immunogenic cell death (ICD) of dying tumor cells and upregulation of the stimulator of interferon genes (STING) is triggered by the generated ROS and Mn2+ from NPMCA , respectively, leading to activation of antitumor immune response. The potency of immune response is further reinforced by reducing the accumulation of adenosine in tumor microenvironment by the activated ADA. As a result, NPMCA enables CDT and SDT-cooperated immunotherapy, showing an obviously improved therapeutic efficacy to inhibit the growths of bilateral tumors, in which the primary tumors are covered with 2-cm tissues.

Boosting B Cell and Macrophage-Mediated Humoral Immunity with Fusion Nanovesicles for Triple-Negative Breast Cancer Combined Therapy.

Cell-derived nanovesicles are widely utilized as therapeutic agents for cancer therapy. Current research mostly focuses on their ability to activate antitumor cellular immunity. However, whether they can activate and participate in antitumor humoral immunity is rarely studied. Here, doxorubicin-loaded hybrid cell nanovesicles (DNVs) are designed for boosting antitumor humoral and cellular immunity. The hybrid cell nanovesicles are generated through fusion of nanovesicles derived from M1-type macrophages and 4T1 tumor cells. It is found that DNVs can accumulate at tumor tissues and draining lymph nodes effectively, which results in the activation of antitumor immune response and significant inhibition of tumor progression. During this process, dendritic cells are effectively activated, subsequently inducing cytotoxicity T lymphocytes-mediated cellular immunity. Furthermore, DNVs elicit the antitumor humoral immunity through boosting T follicular helper cells and germinal center B cells. By analyzing the mechanism behind humoral immunity activation, it is found that M1-type macrophages repolarized by DNVs play an important role. In general, besides antitumor cellular immunity, the proposed hybrid nanovesicles provide a promising strategy for enhancing antitumor humoral immunity by macrophages repolarization and germinal center B cells activation.

Role of Circulating Myeloid-Derived Suppressor Cell (MDSC) in Hodgkin Lymphoma (HL) Progression: Highlighting Clinical Relevance

Introduction. About 15-25% of patients with HL have primary refractory disease or relapse after the first line of therapy and about half of them diagnosed chemosensitivity after transplantation. In the attempt to find a peripheral blood biological marker that could mirror the dysregulated microenvironment of HL, we analyzed the number of myeloid-derived suppressor cells (MDSC) and its correlation with clinical variables and response to therapy. Methods. 52 primary HL patients, (median age: 40, range: 19-61 years; male: 21, female: 31), were included in the study since April, 2018. 63.5% and 36.5% of patients have been diagnosed with early and advanced stages of HL, respectively, and received the ABVD or BEACOPP (14/esc) as a 1st-line treatment regimen. We have also used a routine protocol of metabolic PET assessment according to Deauville criteria of treatment response. Results. 82.6% (43/52) of patients achieved remission (CR/PR) during the follow-up period (median timeline: 25 months). We recorded a disease progression in 6/52 (11.5%) patients during and after the 1st line therapy, (median time to relapse - 8 months). We observed that the level of E-MDSC in patients with HL before treatment is twelve times higher than in healthy control group (24% vs 2 % respectively, p=0.02). Also, the E-MDSC count increases significantly with age of HL patient (with the highest levels in patients over 40 y.o., p=0.008) and with the stage of the disease. Thus patients with advanced stages (III-IV) of HL have increased % of circulating E-MDSC compared to early stages (1A-2B) of disease (p=0.07) . We also found the significant correlation between increased level of MDSC during the treatment and the presence of 4 or more risk factors by IPS (p=0.03). Patients with negative PET results (interim and EOT) had a significantly lower percent of E-MDSC in peripheral blood during and in the end of treatment compare to positive PET patients (interim MDSC: 14% vs 36%, p=0.03; EOT MDSC: 12% vs 42%, p=0.0007, respectively). Patients with CR also had increased number of T and NK-T cells compared to pre-treatment numbers (p=0.05), which all together indicates the activation of anti-tumor immune response. Conclusion. Increased level of circulating E-MDSC is associated with age, advanced stage of HL, presence of 4 or more IPS risk factors and poor clinical outcome. Evaluation of MDSC levels in peripheral blood can be used as additional prognostic marker for early clinical outcome assessment. The discovery of new prognostic factors will improve the individualized approach to the treatment of patients and reduce the risk of disease recurrence.

Sub-Cutaneous Administration of Rituximab Triggers a Pro-Phagocytic Phenotype of Monocytic Compartment in Low-Tumor Burden Follicular Lymphoma Patients Included in the Flirt Clinical Trial, a Lysa Study

Introduction FLIRT (NCT02303119) is a phase III randomized clinical trial designed to evaluate whether Rituximab (R) induction by one IV infusion followed by 3 sub-cutaneous (SC) infusions and short maintenance using SC route (SC-arm) could improve PFS compared to 4-weekly IV R infusions without maintenance (IV-arm) in first line/low tumor burden (GELF criteria) Follicular Lymphoma (FL) patients (202 patients). This study met its primary endpoint and demonstrated that the SC-arm improves PFS of patients with low-tumor burden (#EHA 2022, #ASCO 2022). R is known to exert its anti-tumor activity through complement activation, antibody-dependent cell cytotoxicity, and antibody-dependent cell phagocytosis (ADCP). An ancillary study of the FLIRT trial planned to explore the differential impacts on immune response in the 2 arms and to identify predictive biomarkers. Methods Peripheral blood cells were analyzed in 39 patients from the SC-arm and 43 patients from the IV-arm before treatment (D1) and at M3, i.e. after 4 R infusions in both arms, by multicolor flow cytometry to evaluate circulating T, B, NK, dendritic, and monocyte subset counts and activation status. Results The 82 patients studied display the same clinical features than the whole cohort and the PFS was significantly longer in the SC-arm than in the IV-arm in this limited cohort (P=0.0059). When focusing only on the differences between the 2 arms, monocytes were differentially affected, with an upregulation at M3 of the Fcγ receptors CD32 and CD64 on the 3 main circulating monocyte subsets (CD14hiCD16lo classical, CD14loCD16hi non classical, and intermediate) exclusively in the SC-arm (P<0.05), whereas the SIRPα phagocytosis inhibitory receptor was significantly upregulated on these 3 monocyte subsets exclusively in the IV-arm (P<0.05). In addition, the percentage of CD25hiFoxp3pos Treg among CD4pos T cells was decreased only in the SC-arm (P<0.01), together with an increase of naïve (CD45RAposCCR7pos) and effector memory (CD45RAnegCCR7neg) CD4pos T cells (P<0.05), and naïve CD8pos T cells (P<0.05). Interestingly, CD32 and CD64 expression on M3 monocytes in the SC-arm was correlated with T-cell activation, as evaluated by the expression of HLA-DR on CD8pos T cells and PD-1 on CD4pos T cells (Spearman, P<0.05). Conversely, no modification of T cell subsets was found exclusively in the IV-arm. Additional studies are ongoing to establish whether this differential impact on monocyte phenotype translate to transcriptomic and functional differences. Conclusions Treatment of low-tumor burden FL with R only using SC route is associated with a specific pro-phagocytic activation profile of the monocytic compartment that is not found when R is administrated exclusively by the IV route that favors conversely an anti-phagocytic polarization. This SC-specific monocyte reprogramming is associated with a T-cell activation phenotype, opening the way for additional studies on the impact of R route of administration on ADCP and anti-tumor immune response activation.