Apoptotic Tumor Cells Research Articles

Engineering nanoliposomes to enhance cancer immunotherapy by reversing immunosuppression and synergistically boosting tumour immunogenicity

The clinical success of immune checkpoint inhibitors (ICIs) has initiated a new era in the treatment of metastatic cancer. Unfortunately, the overall response rate of ICIs in pretreated metastatic breast cancer (BC) is generally less than 20% due to many challenges, including low immunogenicity and immunosuppressive tumour microenvironment (ITM). Herein, we synthesized nanoliposomes that codelivered diclofenac (DC) and Mn (5,10,15,20-tetrakis (4-chlorophenyl) porphyrin) Cl (PMnCl) (denoted MD@Lip) to overcome these challenges. DC could block lactate transporters (monocarboxylate transporters 1 and 4) on the cancer cell membrane, thus reducing the concentration of lactate, which acts as a critical regulator of the ITM. Meanwhile, PMnCl-induced sonodynamic therapy (SDT) could induce apoptotic and/or necrotic tumour cell death. Importantly, SDT could induce immunogenic cell death (ICD) during the tumour cell debris generation, which changed the state of tumour cells from nonimmunogenic to immunogenic. Thus, MD@Lip was verified to activate T cells, inactivate regulatory T cells and regulate the levels of immune cytokines, ultimately inhibiting primary and distant tumours by improving the antitumour effect of anti-PD1-based immunotherapy.

T-lymphocytes from focused ultrasound ablation subsequently mediate cellular antitumor immunity after adoptive cell transfer immunotherapy.

Our previous studies found that high-intensity focused ultrasound (HIFU) stimulated tumor-specific T cells in a mouse H22 tumor model, and adoptive transfer of the T cells from HIFU-treated mice could subsequently elicit stronger inhibition on the growth and progression of the implanted tumors. The aim of this study was to investigate the mechanism of T cells from focused ultrasound ablation in HIFU-mediated immunomodulation. Sixty H22 tumor-bearing mice were treated by either HIFU or sham-HIFU, and 30 naïve syngeneic mice served as controls. All mice were euthanized on day 14 after HIFU and splenic T cell suspensions were obtained in each group. Using an adoptive cell transfer model, a total of 1 × 106 T cells from HIFU treated-mice were intravenously injected into each syngeneic H22 tumor-bearing mouse twice on day 3 and 4, followed by the sacrifice for immunological assessments at 14 days after the adoptive transfer. T cells from HIFU-treated mice could significantly enhance the cytotoxicity of CTLs (p < 0.001), with a significant increase of TNF-α (p < 0.001) and IFN-γ secretion (p < 0.001). Compared to control and sham-HIFU groups, the number of Fas ligand+ and perforin+ tumor-infiltrating lymphocytes (TILs) and apoptotic H22 tumor cells were significantly higher (p < 0.001) in the HIFU group. There were linear correlations between apoptotic tumor cells and Fas ligand+ TILs (r = 0.9145, p < 0.001) and perforin+ TILs (r = 0.9619, p < 0.001). T cells from HIFU-treated mice can subsequently mediate cellular antitumor immunity, which may play an important role in the HIFU-based immunomodulation.

Distinct role of CD8 cells and CD4 cells in antitumor immunity triggered by cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir system.

Immune cells can recognize tumor-associated antigens released from dead tumor cells, which elicit immune responses, potentially resulting in tumor regression. Tumor cell death induced by chemotherapy has also been reported to activate immunity. However, various studies have reported drug-induced immunosuppression or suppression of inflammation by apoptotic cells. Thus, this study aimed to investigate whether apoptotic tumor cells trigger antitumor immunity independent of anticancer treatment. Local immune responses were evaluated after direct induction of tumor cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system. The inflammatory response was significantly altered at the tumor site after apoptosis induction. The expression of cytokines and molecules that activate and suppress inflammation simultaneously increased. The HSV-tk/GCV-induced tumor cell apoptosis resulted in tumor growth suppression and promoted T lymphocyte infiltration into tumors. Therefore, the role of T cells after inducing tumor cell death was explored. CD8 T cell depletion abrogated the antitumor efficacy of apoptosis induction, indicating that tumor regression was mainly dependent on CD8 T cells. Furthermore, CD4 T cell depletion inhibited tumor growth, suggesting the potential role of CD4 T cells in suppressive tumor immunity. Tumor tissues were evaluated after tumor cell apoptosis and CD4 T cell depletion to elucidate this immunological mechanism. Foxp3 and CTLA4, regulatory T-cell markers, decreased. Furthermore, arginase 1, an immune-suppressive mediator induced by myeloid cells, was significantly downregulated. These findings indicate that tumors accelerate CD8 T cell-dependent antitumor immunity and CD4 T cell-mediated suppressive immunity. These findings could be a therapeutic target for immunotherapy in combination with cytotoxic chemotherapy.

Caspase-3-Responsive Fluorescent/Photoacoustic Imaging of Tumor Apoptosis.

Caspase-3 is an essential executor in apoptosis, and its activation has been regarded as a biomarker of cell apoptosis. The development of Caspase-3-responsive multimodal probes is a promising research prospect. Fluorescent/photoacoustic (FL/PA) imaging has attracted considerable attention due to the high sensitivity of FL as well as the high spatial resolution and penetration depth of PA. To our knowledge, there has been no tumor-targeted FL/PA probe to monitor the activity of Caspase-3 in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Bio-DEVD-HCy) for Caspase-3-responsive imaging of tumor apoptosis. Ac-DEVD-HCy without tumor-targeted biotin is used as a control probe. In vitro experiments indicated that Bio-DEVD-HCy is superior to Ac-DEVD-HCy because of the higher kinetic parameter of Bio-DEVD-HCy in comparison to Ac-DEVD-HCy. Cell and tumor imaging results suggested that Bio-DEVD-HCy could enter and accumulate in tumor cells with higher FL/PA signal with the help of tumor-targeted biotin. In detail, Bio-DEVD-HCy or Ac-DEVD-HCy could image apoptotic tumor cells with 4.3-fold or 3.5-fold FL enhancement and 3.4-fold or 1.5-fold PA enhancement. Bio-DEVD-HCy or Ac-DEVD-HCy could image tumor apoptosis with 2.5-fold or 1.6-fold FL enhancement and 4.1-fold or 1.9-fold PA enhancement. We envision that Bio-DEVD-HCy will be applied for FL/PA imaging of tumor apoptosis in clinical settings.

Bioengineered Nanospores Selectively Blocking LC3-Associated Phagocytosis in Tumor-Associated Macrophages Potentiate Antitumor Immunity.

Although cytotoxic treatments hold tremendous potential in boosting antitumor immunity, efferocytosis of tumor-associated macrophages (TAMs) could negatively remove apoptotic tumor cells through LC3-associated phagocytosis (LAP), resulting in inefficient tumor antigen presentation and immunosuppressive tumor microenvironment. To address this issue, we developed TAM-targeting nanospores (PC-CW) inspired by the predominant tropism of Rhizopus oryzae toward macrophages. To construct PC-CW, we disguised poly(sodium-p-styrenesulfonate) (PSS)-coated polyethylenimine (PEI)-shRNA nanocomplexes with the cell wall of R. oryzae conidia. LAP blockade by PC-CW delayed the degradation of engulfed tumor debris within TAMs, which not only enhanced antigen presentation but also initiated the domino effect of the antitumor immune response through STING signaling and TAM repolarization. Benefiting from this, PC-CW successfully sensitized the immune microenvironment and amplified CD8+ T cell responses following chemo-photothermal therapy, leading to substantial tumor growth control and metastasis prevention in tumor-bearing mouse models. The bioengineered nanospores represent a simple and versatile immunomodulatory strategy targeting TAMs for robust antitumor immunotherapy.

The effect of the ethanol extract of Typhonium flagelliforme on apoptosis adenocarcinoma mamma cells in C3H mice

Malignancy cause high impact in mortality in worldwide. Typhonium Flagelliforme have been known to improve the immune system. The aims of this study was to determine the effects of Typhonium Flagelliforme on the tumor necrosis factor alpha (TNF-α) level and percentage of apoptotic cells in mice tumor The post test study design with randomized control group of 24 C3H mice with tumor were divided into 4 groups: I (control), 2 ; 3; and 4 (treatment). All treatment were given 0.2 cc of extract at the dose of 200, 400 and 800 mg/kgBW respectively for 30 days. TNF-α level was from tumor tissue were measured using ELISA methods, and percentage of apoptotic cells from tumor tissue were determined using immunohistochemistry assay with TUNEL antibody. The mean difference of TNF-α expression was tested using Anova at the significant level of 0.05. Apoptotic cells was analyzed by Mann Whitney. The result indicate that average of TNF α level in the treated groups were higher than those of control group (p&lt;0.05). The percentage of apoptotic cells at the dose 200 mg/KgBW was higher than that of control group, groups 2 and 3(P&lt;0.05). In conclusion, Typhonium Flagelliforme can increase in the percentage of apoptosis cells but not in the levels of TNF α Bangladesh Journal of Medical Science Vol. 22 No. 02 April’23 Page : 329-335

Abstract 1272: Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) has risen to become the 3rd leading cause of cancer related deaths in the United States with a 5-year survival rate of 11% and a median survival of just 4-6 months. PDA is notoriously chemo-resistant and has shown little to no sensitivity to the current immune checkpoint blockade therapy. A novel marker for the tumor microenvironment is phosphatidylserine, first characterized as a selective marker of tumor vasculature. Phosphatidylserine (PS) is a phospholipid exposed on the outer leaflet of the plasma membrane of tumor associated endothelial cells, apoptotic tumor cells, and some viable tumor cells, where it functions to suppress the immune response. Exposed PS drives immunosuppression by binding to PS receptors expressed on tumor-infiltrating macrophages and dendritic cells. To this point, PS has been targeted with antibodies, such as Bavituximab, that have shown excellent specificity for tumor vasculature and an immune stimulatory environment evidence by polarization of macrophages to a pro-inflammatory M1-like phenotype, a reduction in myeloid-derived suppressor cells, enhanced maturation of dendritic cells and increased primed T cell activity. We have advanced this concept by developing the next generation of PS targeting agent, a fusion protein (betabody) that exploits the PS-binding domain of β2-glycoprotein 1 (β2GP1), an abundant serum protein that binds PS. Betabodies were constructed by fusing domain V of β2GP1 to the Fc of an IgG2a. Betabodies are smaller than full length antibodies (~85 kDa vs 150 kDa), have good affinity for PS (~1 nM) and bind directly to PS. We have demonstrated that betabodies bind specifically to externalized PS through ELISA, ICC and flow cytometry. We established that betabodies localize robustly and specifically to the tumor microenvironment through in vivo localization experiments in tumor-bearing mice. Further studies will exploit the specificity of betabodies to deliver novel microtubule-disrupting agents to exposed PS in the microenvironment of PDA. Because betabodies are internalized inefficiently, therapeutic payloads will be linked to betabodies via enzyme cleavable linkers. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors. In addition, similarly to PS targeting antibodies, preliminary data in vitro has shown that treatment of both bone marrow derived macrophages and Raw 264.7 with the unconjugated betabodies repolarize M2 macrophages to a M1 phenotype. The mechanism of action has not yet been fully elucidated; however, it may involve the interaction between the β2GP1 domains and endogenous LPS. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors via targeted drug delivery as well as modulation of the immuno-microenvironment like its PS targeting predecessors. Citation Format: Natalie Zara Phinney, Xianming Huang, Mary Lynn Trawick, Kevin Pinney, Rolf Brekken. Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer [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 1272.

Abstract 6389: Differential role of CD8 cells and CD4 cells in anti-tumor immunity triggered by tumor cell apoptosis using HSVtk/GCV system

Abstract Immune cells can recognize tumor antigens and elicit an adaptive immune response, potentially resulting in tumor regression. Drug-induced dead tumor cells have been reported to enhance anti-tumor immunity, but results are conflicting, possibly due to the immunosuppressive effects of the cytotoxic drugs. To investigate whether apoptotic tumor cells trigger anti-tumor immunity independent of anti-cancer treatment, local immune responses were evaluated after direct induction of tumor cell apoptosis using a herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system. The inflammatory cytokines IFNγ and IL-12β and anti-inflammatory factor PD-1 and TGFβ1 were upregulated at the tumor site after apoptosis induction. HSV-tk/GCV-induced tumor cell apoptosis resulted in suppression of tumor growth in vivo and promoted the infiltration of CD8 and CD4 T lymphocytes in tumor. To explore the role of T cells after induction of tumor cell death, GCV was administered together with anti-CD8 or anti-CD4 depleting antibodies to syngeneic HSV-tk transfected tumor-bearing mice. CD8 T cell depletion abrogated the anti-tumor efficacy of GCV, indicating that tumor regression was mainly dependent on CD8 T cells. This anti-tumor immunity was enhanced by anti-PD-1 antibody injection. On the contrary, CD4 T cell depletion further inhibited tumor growth than GCV alone, suggesting the potential role of CD4 T cells in suppressive tumor immunity. To elucidate this immunological mechanism, tumor tissues were evaluated after tumor cell apoptosis and CD4 T cell depletion. Foxp3, a regulatory T cell marker, was decreased after CD4 T cell depletion. In addition, Arg1, a marker of immunosuppressive myeloid cells, was significantly downregulated, which was expressed on tumor-associated macrophages (TAMs) and monocytic myeloid-derived suppressor cells (mMDSCs). Collectively, these findings indicate that tumor cell apoptosis accelerates CD8 T cell-dependent anti-tumor immunity and CD4 T cell-mediated Arg1 production from TAMs and mMDSCs. Citation Format: Sho Umegaki, Hidekazu Shirota, Tomoyuki Iwasaki, Yuki Kasahara, Chikashi Ishioka. Differential role of CD8 cells and CD4 cells in anti-tumor immunity triggered by tumor cell apoptosis using HSVtk/GCV system [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 6389.

Abstract 4011: Preferential cytotoxicity of ESK981 in neuroendocrine prostate cancer

Abstract Introduction: Despite advances in the understanding of neuroendocrine prostate cancer (NEPC) development, effective therapeutic options remain limited. We previously reported that ESK981, a phase I-cleared multi-tyrosine kinase inhibitor (MTKI), exhibited tumor growth inhibitory abilities in multiple preclinical castration resistant prostate cancer (CRPC) and androgen-negative models by blocking the PIKfyve activity and disrupting autophagy. The results suggested that AR-negative prostate cancer have better response to ESK981 induced tumor inhibition than AR+ prostate cancer (Qiao et al, Nature Cancer 2022). Methods: In order to examine the tumor growth efficacy of ESK981 in NEPC, we assessed the efficacy of ESK981 monotherapy in six NEPC (LTL-352, LTL-331R, LTL-545, and LTL-610 patient-derived xenografts (PDXs); and NCI-H660 cell line-derived xenograft) in vivo models. PDXs were maintained in CB17 SCID male mice and passaged by subcutaneous implantation. When the tumors reached ~100mm3, mice were randomized and treated with either vehicle (Ora-plus) or 30mg/kg ESK981 administered by oral gavage once a day in a five-day per week schedule. Mouse body weight and tumor volume was monitored throughout the treatment schedule. Subcutaneous tumors were collected post five days of treatment for early assessment and long-term treatment for efficacy evaluation, respectively. H&amp;E staining, TUNEL in situ cell death assay, and western blotting were performed to determine the morphology changes and apoptosis after ESK981 treatment. Single cells were isolated from tumors and stained with Zombie live/dead dye, CD45, CD11b and Ly6G fluorescently conjugated antibodies to assess neutrophil population by flow cytometry. Conclusions: We demonstrated that ESK981 monotherapy is well tolerated in all in vivo models and ESK981 exerted greater cytotoxicity in NEPC than previous evaluated prostate adenocarcinoma by percent tumor growth inhibition. ESK981 also induced dramatic cell deaths in NEPC preclinical models, determined by H&amp;E staining, TUNEL-positive apoptotic tumor cells, and increased protein level of PARP cleavage via western blotting. Moreover, flow cytometry analysis revealed a dramatic increase in the intratumoral neutrophil infiltration after ESK981 treatment in the NEPC preclinical models in a time-dependent manner. NEPC tumors are sensitive to ESK981 monotherapy in vivo, suggesting NEPC patients may be the right population to target with ESK981. Together, ESK981 monotherapy is safe and effective therapy in NEPC preclinical models, and these results will warrant the clinical trial of ESK981 on NEPC patients. Citation Format: Yang Zheng, Kyle J. Garcia-Rogers, Yi Bao, Rahul Mannan, Tongchen He, Sarah N. Yee, Caleb Cheng, Xia Jiang, Isabella E. Taylor, Xuhong Cao, Yuzhuo Wang, Yuanyuan Qiao, Arul M. Chinnaiyan. Preferential cytotoxicity of ESK981 in neuroendocrine prostate cancer. [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 4011.

Colorectal cancer-derived extracellular vesicles containing HSP70 enhance macrophage phagocytosis by up-regulating MARCO expression

In recent years, we have realized that extracellular vesicles (EVs) play a critical role in regulating the intercellular communication between tumor and immune cells in the tumor microenvironment (TME). Tumor-derived extracellular vesicles (TDEVs) profoundly affect the functional changes of tumor-associated macrophages (TAMs) and promote their M2 polarization. Meanwhile, macrophages have a strong phagocytic ability in phagocytosing apoptotic cells. Especially in the course of chemotherapy or radiotherapy, TAMs can phagocytose and remove apoptotic tumor cells, showing anti-inflammatory and pro-tumor effects. However, the underlying mechanisms by which TDEVs regulate macrophage phagocytosis of apoptotic tumor cells have not been fully elucidated. In this study, we focused on the effect of colorectal cancer-derived extracellular vesicles (CRC-EVs) on macrophages. We demonstrated that CRC-EVs enhanced macrophage phagocytosis of apoptotic CRC cells. We then determined that heat shock protein 70 (HSP70) carried in CRC-EVs was responsible for this effect by using mass spectrometry-based proteomic analysis and the CRISPR-Cas9 system. Through transcriptome sequencing of macrophages, we found that the enhanced phagocytosis of macrophages was mainly due to the up-regulation of the macrophage receptor with collagenous structure (MARCO). In addition, we confirmed that the up-regulation of MARCO was mediated by the AKT-STAT3 signaling pathway. Taken together, this study revealed a novel EVs-mediated macrophage phagocytosis mechanism involved in the clearance of apoptotic tumor cells in the TME. Targeting TDEVs may have potential therapeutic applications in tumor treatment.

Preclinical PET Imaging of Tumor Cell Death following Therapy Using Gallium-68-Labeled C2Am

There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.

Phytoradiotherapy to enhance cancer treatment outcomes with cannabidiol, bitter melon juice, and plant hemoglobin.

Despite technological advances in radiation therapy for cancer treatment, many patient populations still experience mediocre survival percentages, local control, and quality of life. Additionally, much of the world lacks access to expensive, modern treatment options. The need for innovative, cost-effective solutions that can improve patient treatment outcomes is essential. Phytomedicines have been shown to induce apoptotic tumor cell death, diminish tumor progression, reduce cancer incidence, alleviate harmful hypoxic conditions, and more. While an ample amount of research is available that characterizes many phytomedicines as having anti-cancer properties that increase tumor cell killing/control and mitigate the harmful side effects of radiation damage, little work has been done to investigate the synergistic effect of phytoradiotherapy: combining radiation treatment with phytomedicines. In this study, a protocol for testing the radiosensitizing effects of phytomedicines was validated and used to investigate the well-known plant based medicine cannabidiol (CBD) and the lesser-known medicinal fruit Bitter Melon. Additionally, based on its high concentration of plant hemoglobin which has been shown to abate hypoxia, the African-indigenous Justicia plant was tested in pancreatic adenocarcinoma mouse models. The studies reveal that these phytomedicines can effectively enhance tumor cell killing, minimize tumor growth, and prolong mice survival. There is certainly the need for additional research in this regard, however, phytoradiotherapy: the use of phytomedicines to enhance radiation therapy treatment outcomes, continues to show potential as a promising, innovative way to improve cancer care.

Killing cancer softly: The resolution of cancer lies in tumor cells

Killing cancer softly: The resolution of cancer lies in tumor cells Dr Dipak Panigrahy, M.D., an Assistant Professor at Harvard Medical School looks at killing tumor cells to resolve the cancer epidemic. For decades, the standard paradigm in cancer has been to kill tumor cells either directly, via cytotoxic chemotherapy, radiation, or target-selective inhibitors, or indirectly through immune-mediated or anti-angiogenic approaches. However, in 1956, Dr. Laszlo Révész first demonstrated that co-injection of irradiated dead cells with tumor cells reduced the number of tumor cells needed to produce tumors in rodents, and accelerated tumor progression (Révész phenomenon) (1). We demonstrated that cytotoxic cancer therapy stimulated cancer growth via cell debris, as our studies are consistent with previous research on radiation-generated apoptotic (dead) tumor cells. Dead-cell-stimulated growth is a natural part of the tissue regeneration cycle, whereas debris is interpreted by the tissue as an injury signal and stimulates wound healing and regeneration. Cancer can be viewed as run-away wound healing and regeneration that fails to be self-limited.

Fabrication of metformin and survivin siRNA encapsulated into polyethyleneimine-altered silk fibroin nanoparticles for the treatment of nasopharyngeal carcinoma

Metformin (MET) and survivin siRNA were delivered using silk fibroin nanoparticles (SFNs) modified with polyethyleneimine (PEI). Nanoparticle functional groups, structural, stability and physicochemical characteristics of the nanoparticles were determined. SFNs loaded with siRNA that had appropriate size, zeta potential, and stability were confirmed by various analytical and spectroscopical methods. The vitro cytotoxicity analysis was performed using CNE2 nasopharyngeal carcinoma cells with different compositions, and the results showed that the newly fabricated nanoparticles effectively triggered apoptosis in CNE2. Afterward, the mice analysis was used to investigate the in vivo therapeutic efficacy. With no apparent weight loss, the co-delivery systems dramatically slowed nasopharyngeal tumor development in mice. Survivin siRNA and MET increased the number of apoptotic tumor cells on histopathology slides. Apoptosis induction in cancer cells and therapeutic effectiveness were satisfactory with the drug delivery method. In this way, it might be used as a therapeutic agent in the fight against nasopharyngeal cancer.

Abstract PR021: CCR1 expression defines pancreatic tumor associated macrophages and drives their immunosuppressive properties

Abstract The tumor microenvironment of pancreatic ductal adenocarcinoma (PDA) includes abundant fibroblasts and infiltrating immune cells, the latter largely immunosuppressive. Immunotherapy approaches have been ineffective in PDA, pointing to the need for a better understanding of the mechanisms of immunosuppression. We previously identified C-C Motif Chemokine Receptor 1 (CCR1) as overexpressed in macrophages exposed to pancreatic cancer cell conditioned medium in vitro. By single-cell RNA sequencing, we found CCR1 to be expressed by tumor associated macrophages (TAMs) and granulocytes in both human and mouse PDA. Conversely, the ligands for CCR1, C-C Motif Chemokine Ligands (CCLs), are produced by tumor associated fibroblasts. Thus, we sought to investigate the functional role of CCR1 in pancreatic cancer using a combination of genetically engineered mouse models and pharmaceutically approaches. In a first set of experiments, we generated KC;CCR1-/- mice (Ptf1a-Cre;LSL-KrasG12D;CCR1-/-) to determine the requirement of CCR1 during oncogenic KRAS induced pancreatic cancer initiation. We did not observe a difference in PanIN formation/progression in KC;CCR1-/- compared to KC mice. However, we observed increased immune infiltration, including CD8 T cells, in KC;CCR1-/- pancreata. In a second set of experiments, we then orthotopically transplanted two independent mouse pancreatic cancer cells derived from the KPC model in syngeneic CCR1 knockout hosts. We observed reduced tumor growth, which was rescued by CD8 T cell depletion, indicating an increase of anti-tumor immunity in mice lacking CCR1. Consistently, we observed elevated cytotoxic Granzyme B expression, as well as an increase of apoptotic cells in tumors harvested from Ccr1-/- mice. Through mass cytometry (CyTOF) and co-immunofluorescence staining we discovered that tumor associated macrophages from CCR1-/- mice expressed less Arginase 1 and CD206, both immunosuppressive markers, compared to macrophages in wild type tumors. In the last set of experiments, we used the CCR1 inhibitor J-113863, administered to mice following establishment of an orthotopically implanted tumor. Similar to the genetic model, CCR1 inhibition resulted in smaller tumors. Further, targeting CCR1 either genetically or using a CCR1 inhibitor synergizes with anti-PDL1 immune checkpoint blockade to reduce tumor growth. Together, our data is consistent with the notion that tumor associated macrophages lacking CCR1 expression are less immunosuppressive, consequently allowing increased CD8 T cell-mediated anti-tumor immunity. Targeting CCR1 in combination with immune checkpoint blockade improves antitumor efficacy in pancreatic cancer. Citation Format: Yaqing Zhang, Wei Yan, Kristee L. Brown, Ashely Velez-Delgado, Zeribe C. Nwosu, Katelyn Donahue, Padma Kadiyala, Sion Yang, Faith R. Avritt, Xi He, Carlos Espinoza, Marina Pasca di Magliano. CCR1 expression defines pancreatic tumor associated macrophages and drives their immunosuppressive properties [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR021.

Abstract C079: Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) has risen to become the 3rd leading cause of cancer related deaths in the United States with a 5-year survival rate of 11% and a median survival of just 4-6 months. PDA is notoriously chemoresistant and has shown little to no sensitivity to the current immune checkpoint blockade therapy. A novel marker for the tumor microenvironment that has emerged is phosphatidylserine, initially characterized as a selective marker of tumor vasculature. Phosphatidylserine (PS) is a phospholipid exposed on the outer leaflet of the plasma membrane of tumor associated endothelial cells, apoptotic tumor cells, and some viable tumor cells, where it functions to suppress the immune response. Exposed PS drives immunosuppression by binding to PS receptors expressed on tumor-infiltrating macrophages and dendritic cells. To this point, PS has been targeted with antibodies, such as Bavituximab, that have shown excellent specificity for tumor vasculature and an immune stimulatory environment evidence by polarization of macrophages to a pro-inflammatory M1-like phenotype, a reduction in myeloid-derived suppressor cells, enhanced maturation of dendritic cells and increased primed T cell activity. We have advanced this concept by developing the next generation of PS targeting agent, a fusion protein (betabody) that exploits the PS-binding domain of β2-glycoprotein 1 (β2GP1), an abundant serum protein that binds PS. Betabodies were constructed by fusing domain V of β2GP1 to the Fc of an IgG2a. Betabodies are smaller than full length antibodies (~85 kDa vs 150 kDa), have good affinity for PS (~1 nM) and bind directly to PS. We have demonstrated that betabodies bind specifically to externalized PS through ELISA, ICC and flow cytometry. We established that betabodies localize robustly and specifically to the tumor microenvironment through in vivo localization experiments in tumor-bearing C57Bl/6 mice. Further studies will exploit the specificity of betabodies to deliver novel microtubule-disrupting agents to exposed PS in the microenvironment of PDA. Due to the fact that betabodies are internalized inefficiently, therapeutic payloads will be linked to betabodies via enzyme cleavable linkers. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors. Citation Format: Natalie Z. Phinney, Xianming Huang, Rolf Brekken. Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C079.

Circulating caspase-cleaved cytokeratin 18 correlates with tumour burden and response to therapy in patients with colorectal cancer liver metastasis

Background and aimsSystemic chemotherapy followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CRCLM) but reliable biomarkers predicting response to therapy are needed. Spontaneous apoptosis of single tumour cells is common in CRCLM. We explored the potential of circulating apoptosis markers to predict treatment response. Materials and methodsFifty-eight patients with CRCLM or hepatocellular carcinoma (HCC) were included in this study. Tumour tissue and blood samples were obtained before and after initiation of chemotherapy. Immunohistochemistry and ELISA assays were utilized to quantify the apoptosis marker caspase-cleaved cytokeratin 18 (M30) in tissue and circulation. ResultsCRCLM tissues showed more apoptotic tumour cells than HCC, or healthy liver. This was associated with elevated levels of circulating M30 (median = 244 U/l vs. 37 U/l in healthy controls, p = 0.009) which correlated with tumour volume (r2 = 0.92). Patients with progressive disease during chemotherapy showed higher M30 levels before therapy than responders (745 U/l vs. 136 U/l, p = 0.016). The predictive potential of M30 was higher than that of the tumour markers CA19-9 or CEA (AUC: 0.93, 0.63, and 0.78, respectively). ConclusionsApoptotic tumour cells release cellular debris into the circulation, which provides information about tumour size and vitality.

Histopathological impact of Redox-responsive methacrylamide based micellar nanoparticles on Orthotopic Models of Triple Negative Breast Cancers

The therapeutic efficacy of anticancer nanocarriers ishighly dependent on their size, shape, targeting ability, andstimuli-responsiveness. Herein, we studied the in vivo therapeutic efficacy ofDoxorubicin (Dox) loaded redox responsive micellar-like nanoparticles (MNPs)based on linear 2-hydroxypropyl methacrylamide (HPMA) via histopathologicalevaluations. The therapeutic efficacy of DOX-loaded micellar-like Nanoparticlessignificantly improved while the side effects reduced as confirmed byhistopathological examinations. H&amp;amp;E and tunnel staining of tumor tissuesindicated the higher population of apoptotic tumor cells in both treatmentgroups containing DOX. These redox responsive crosslinked&amp;nbsp;HPMA-basedmicellar-like nanoparticles with acceptable therapeutic efficacy and apoptosisinduction in cancerous cells proved to be promising nanomedicine for breast cancer chemotherapy.

Mobilizing phospholipids on tumor plasma membrane implicates phosphatidylserine externalization blockade for cancer immunotherapy.

In "healthy" tumor cells, phosphatidylserine (PS) is predominately localized in the inner plasma membrane leaflet. During apoptosis, PS relocates to the outer leaflet. Herein, we established PSout tumor models with tumor cells lacking PS flippase component CDC50A, constantly exposing PS but alive. PSout tumors developed bigger than wild-type (WT) tumors, featuring M2 polarized tumor-associated macrophages (TAMs) and fewer tumor-antigen-specific Tcells. The PS receptor TIM-3 is responsible for PS recognition. Employing an opposite tumor model, PSin, with tumor cells lacking the PS scramblase Xkr8 and unable to expose PS during otherwise normal apoptosis, we find that the accumulated apoptotic tumor cells produce and release cyclic GAMP (cGAMP) to immune cells to activate the STING pathway, leading to TAM M1 polarization, suppressed interleukin (IL)-10 secretion, and natural killer (NK) cell cytotoxicity. Silencing Xkr8 invivo by either short hairpin RNA (shRNA) or small interfering RNA (siRNA) to achieve a PS externalization blockade provides robust therapeutic anti-tumor efficiency.

Patient-derived organoids potentiate precision medicine in advanced clear cell renal cell carcinoma.

To investigate the role of patient-derived organoid (PDO) model in the precision medicine of advanced clear cell renal cell carcinoma (ccRCC), we retrospectively analyzed the clinical data of seven cases of ccRCC diagnosed by operation and pathology in Renji Hospital from September 2021 to September 2022. The seven patients were diagnosed with advanced ccRCC with or without remote metastasis. Cytoreductive and radical nephrectomy was performed respectively. To predict the response to immunotherapy and provide personalized medicine recommendation, a PDO model based on air-liquid interface system was established from the surgical resected tumor and subsequent drug screening was performed. Hematoxylin and eosin (H&E) staining and immunohistochemistry revealed that the PDO recapitulated the histological feature of parent tumor. Immunofluorescence staining identified that CD3+ T cells, SMA+ cancer associated fibroblasts, and CD31+ endothelial cells were preserved in PDO models. Fluorescence activated cell sorter (FACS) revealed an evidently increased ratio of CD8+/CD4+ T cells and apoptotic tumor cells in PDO treated with toripalimab than those treated with IgG4. The results showed that toripalimab is able to rescue the excessive death of CD8+ T cells by critically reversing the immune exhaustion state of ccRCC in PDO model. This research validated that PDO is a promising and faithful preclinical model for prediction of immunotherapy response in patients with ccRCC.