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International Journal of Molecular... Jun 2024V-set immunoglobulin domain-containing 4 (VSIG4) is a B7 family protein with known roles as a C3 fragment complement receptor involved in pathogen clearance and a...
Antibodies Targeting Human or Mouse VSIG4 Repolarize Tumor-Associated Macrophages Providing the Potential of Potent and Specific Clinical Anti-Tumor Response Induced across Multiple Cancer Types.
V-set immunoglobulin domain-containing 4 (VSIG4) is a B7 family protein with known roles as a C3 fragment complement receptor involved in pathogen clearance and a negative regulator of T cell activation by an undetermined mechanism. VSIG4 expression is specific for tumor-associated and select tissue-resident macrophages. Increased expression of VSIG4 has been associated with worse survival in multiple cancer indications. Based upon computational analysis of transcript data across thousands of tumor and normal tissue samples, we hypothesized that VSIG4 has an important role in promoting M2-like immune suppressive macrophages and that targeting VSIG4 could relieve VSIG4-mediated macrophage suppression by repolarizing tumor-associated macrophages (TAMs) to an inflammatory phenotype. We have also observed a cancer-specific pattern of VSIG4 isoform distribution, implying a change in the functional regulation in cancer. Through a series of in vitro, in vivo, and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages. Across patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment resulted in the upregulation of cytokines associated with TAM repolarization and T cell activation and chemokines involved in immune cell recruitment. VSIG4 blockade is also efficacious in a syngeneic mouse model as monotherapy as it enhances efficacy in combination with anti-PD-1, and the effect is dependent on the systemic availability of CD8 T cells. Thus, VSIG4 represents a promising new target capable of triggering an anti-cancer response via multiple key immune mechanisms.
Topics: Animals; Humans; Mice; Tumor-Associated Macrophages; Neoplasms; Cell Line, Tumor; Lymphocyte Activation; Mice, Inbred C57BL; Cytokines; Female; Receptors, Complement
PubMed: 38892347
DOI: 10.3390/ijms25116160 -
Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation.Cells May 2024Several chronic inflammatory diseases have been linked to high-salt (HS) diets. Chronic inflammation is an established causative hallmark of cancer. However, a direct...
Several chronic inflammatory diseases have been linked to high-salt (HS) diets. Chronic inflammation is an established causative hallmark of cancer. However, a direct role of HS diets in tumorigenesis is yet to be defined. Previous orthotopic murine breast tumor studies have shown that short-term HS diets caused inhibition of tumor growth through the activation of cytotoxic adaptive immune responses. However, there have been experimental challenges in developing a viable chronic HS-diet-based murine tumor model. To address this, we have developed a novel chronic HS diet tumor model through the sequential passaging of tumor cells in mice under HS dietary conditions. Two orthotopic murine triple-negative breast cancer models, 4T1 tumor cells injected into BALB/c mice and Py230 tumor cells injected into C57Bl/6 mice, were utilized in our study. For the HS diet cohort, prior to orthotopic injection with tumor cells, the mice were kept on a 4% NaCl diet for 2 weeks. For the regular salt (RS) diet cohort, the mice were kept on a 1% NaCl diet. Following syngeneic cancer cell injection, tumors were allowed to grow for 28 days, following which they were collected to isolate immune cell-depleted cancer cells (passage 1, P1). The tumor cells from P1 were reinjected into the next set of non-tumor-bearing mice. This procedure was repeated for three cycles (P2-P4). In P1, compared to the RS diet cohort, we observed reduced tumor kinetics in both murine tumor models on the HS diet. In contrast, by P4, there was significantly higher tumor progression in the HS diet cohort over the RS diet cohort. Flow cytometry analysis demonstrated an 8-fold increase in tumor-initiating stem cells (TISCs) from P1 to P4 of the HS diet cohort, while there were no significant change in TISC frequency with sequential passaging in the RS diet cohort. Molecular studies showed enhanced expression of TGFβR2 and CD80 on TISCs isolated from the P4 HS diet cohort. In vitro studies demonstrated that TGFβ stimulation of these TISCs increased the cellular expression of CD80 molecules. Further, the chronic HS diet selectively induced the glycolytic metabolic phenotype over the mitochondrial oxidative phosphorylation phenotype in TISCs, which is needed for the production of metabolites during tumor cell differentiation and proliferation. The infiltrating CD8 and CD4 T-lymphocytes in P4 tumors demonstrated increased expression of the immune checkpoint inhibitor (ICI) CTLA4, a known binding partner of CD80, to cause immune exhaustion and pro-tumorigenic effects. Interestingly, anti-TGFβ monoclonal antibodies (mAbs) played a synergistic role in further enhancing the anti-tumor effect of anti-CTLA4 mAb. In summary, our findings demonstrated that chronic HS diet increased the frequency of TISCs in tumors leading to blunting of cytotoxic adaptive immune responses causing tumor proliferation. Furthermore, a combination of anti-TGFβ with current ICI-based immunotherapies could exert more favorable anti-cancer clinical outcomes.
Topics: Animals; Female; Neoplastic Stem Cells; Cell Proliferation; Mice; Mice, Inbred BALB C; Cell Line, Tumor; Breast Neoplasms; Sodium Chloride, Dietary; Mice, Inbred C57BL; Humans
PubMed: 38891044
DOI: 10.3390/cells13110912 -
Cannabis and Cannabinoid Research Jun 2024Glioblastoma patients have a highly immunosuppressive tumor microenvironment and systemic immunosuppression that comprise a major barrier to immune checkpoint therapy....
Endocannabinoid Receptor 2 Function is Associated with Tumor-Associated Macrophage Accumulation and Increases in T Cell Number to Initiate a Potent Antitumor Response in a Syngeneic Murine Model of Glioblastoma.
Glioblastoma patients have a highly immunosuppressive tumor microenvironment and systemic immunosuppression that comprise a major barrier to immune checkpoint therapy. Based on the production of endocannabinoids by glioblastomas, we explored involvement of endocannabinoid receptor 2 (CB2R), encoded by the CNR2 gene, which is predominantly expressed by immune cells, in glioblastoma-related immunosuppression. Bioinformatics of human glioblastoma databases was used to correlate enzymes involved in the synthesis and degradation of endocannabinoids, as well as CB2Rs, with patient overall survival. Intrastriatal administration of luciferase-expressing, murine GL261 glioblastoma cells was used to establish in in vivo glioblastoma model for characterization of tumor growth and intratumoral immune cell infiltration, as well as provide immune cells for in vitro co-culture experiments. Involvement of CB2Rs was determined by treatment with CB2R agonist (GW405833) or CB2R antagonist (AM630). ELISA, FACS, and immunocytochemistry were used to determine perforin, granzyme B, and surface marker levels. Bioinformatics of human glioblastoma databases showed high expression of CB2R and elevated endocannabinoid production correlated with poorer prognosis, and involved immune-associated pathways. AM630treatment of GL261 glioblastoma-bearing mice induced a potent antitumor response, with survival plateauing at 50% on Day 40, when all control mice (median survival 28 days) and mice treated with GW405833 (median survival 21 days) had died. Luciferase tumor imaging revealed accelerated tumor growth by GW405833 treatment, but stable or regressing tumors in AM630-treated mice. Notably, in spleens, AM630 treatment caused an 83% decrease in monocytes/macrophages, and 1.8- and 1.6-fold increases in CD8+ and CD4+ cells, respectively. Within tumors, there was a corresponding decrease in tumor-associated macrophages (TAMs) and increase in CD8+ T cells. In vitro, lymphocytes from AM630-treated mice showed greater cytotoxic function (increased percentage of perforin- and granzyme B-positive CD8+ T cells). These results suggest that inhibition of CB2R enhances both immunosuppressive TAM infiltration and systemic T-cell suppression through CB2R activation, and that inhibition of CB2Rs can potently counter both the immunosuppressive tumor microenvironment, as well as systemic immunosuppression in glioblastoma.
PubMed: 38888628
DOI: 10.1089/can.2024.0063 -
Advanced Science (Weinheim,... Jun 2024Bacteria can be utilized for cancer therapy owing to their preferential colonization at tumor sites. However, unmodified non-pathogenic bacteria carry potential risks...
Bacteria can be utilized for cancer therapy owing to their preferential colonization at tumor sites. However, unmodified non-pathogenic bacteria carry potential risks due to their non-specific targeting effects, and their anti-tumor activity is limited when used as monotherapy. In this study, a biohybrid-engineered bacterial system comprising non-pathogenic MG1655 bacteria modified with CDH17 nanobodies on their surface and conjugated with photosensitizer croconium (CR) molecules is developed. The resultant biohybrid bacteria can efficiently home to CDH17-positive tumors, including gastric, pancreatic, and colorectal cancers, and significantly suppress tumor growth upon irradiation. More importantly, biohybrid bacteria-mediated photothermal therapy (PTT) induced abundant macrophage infiltration in a syngeneic murine colorectal model. Further, that the STING pathway is activated in tumor macrophages by the released bacterial nucleic acid after PTT is revealed, leading to the production of type I interferons. The addition of CD47 nanobody but not PD-1 antibody to the PTT regimen can eradicate the tumors and extend survival. This results indicate that bacteria endowed with tumor-specific selectivity and coupled with photothermal payloads can serve as an innovative strategy for low-immunogenicity cancers. This strategy can potentially reprogram the tumor microenvironment by inducing macrophage infiltration and enhancing the efficacy of immunotherapy targeting macrophages.
PubMed: 38888519
DOI: 10.1002/advs.202401905 -
Journal of Microscopy Jun 2024Breast cancer is one of the leading causes of mortality among women. The tumour microenvironment, consisting of host cells and extracellular matrix, has been...
Breast cancer is one of the leading causes of mortality among women. The tumour microenvironment, consisting of host cells and extracellular matrix, has been increasingly studied for its interplay with cancer cells, and the resulting effect on tumour progression. While the breast is one of the most innervated organs in the body, the role of neurons, and specifically sensory neurons, has been understudied, mostly for technical reasons. One of the reasons is the anatomy of sensory neurons: sensory neuron somas are located in the spine, and their axons can extend longer than a meter across the body to provide innervation in the breast. Next, neurons are challenging to culture, and there are no cell lines adequately representing the diversity of sensory neurons. Finally, sensory neurons are responsible for transporting several different types of signals to the brain, and there are many different subtypes of sensory neurons. The subtypes of sensory neurons, which innervate and interact with breast tumours, are unknown. To establish the tools for labelling and subtyping neurons that interact with breast cancer cells, we utilised two retrograde tracer's standards in neuroscience, wheat-germ agglutinin (WGA) and cholera toxin subunit B (CTB). In vitro, we employed primary sensory neurons isolated from mouse dorsal root ganglia, cultured in a custom-built microfluidic device DACIT, that mimics the anatomical compartmentalisation of the sensory neuron's soma and axons. In vivo, we utilised both syngeneic and transgenic mouse models of mammary carcinoma. We show that CTB and WGA trace different but overlapping sensory neuronal subpopulations: while WGA is more efficient in labelling CGRP+ neurons, CTB is superior in labelling the NF200+ neurons. Surprisingly, both tracers are also taken up by a significant population of breast cancer cells, both in vitro and in vivo. In summary, we have established methodologies for retrograde tracing of sensory neurons interacting with breast cancer cells. Our tools will be useful for future studies of breast tumour innervation, and development of therapies targeting breast cancer-associated neuron subpopulations of sensory neurons. Lay description: Breast cancer is an aggressive disease that affects both women and men throughout the world. While it has been reported that the increasing size of nerves in breast cancer correlates to bad prognosis in patients, the role of nerves, especially sensory nerves, in breast cancer progression, has remained largely understudied. Sensory nerves are responsible for delivering signals such as pain, mechanical forces (pressure, tension, stretch, touch) and temperature to the brain. The human body is densely innervated, and nerves extending into peripheral organs can be as long as a few meters. Nerve classification and function can be very complex, as they contain bundles of extensions (axons) originating in different neuronal bodies (soma). Maintaining neurons and growing axons in cell culture conditions in order to mimic innervation is technically challenging, as it involves multiple organs of the human body. Here, we focus on tracing sensory axons from the breast tumours back to the neuronal soma, located in the dorsal root ganglia, inside the spine. To do so, we are using two different 'retrograde' tracers, WGA and CTB, which are proteins with a natural ability to enter axons and travel in a retrograde fashion, arriving at the soma, even if it means to travel distances longer than a meter. Both tracers are fluorescently labelled, making them visible using high-resolution fluorescent microscopy. We show that both WGA and CTB can label sensory neurons in tumours, or in cell culture conditions. The two tracers differ in efficiency of tracing different sensory neurons subpopulations: while WGA is more efficient in tracing small C-fibres (CGRP-positive), CTB is more efficient in tracing A-fibres (NF200+) of sensory neurons. In summary, we have successfully established retrograde tracing techniques for sensory neurons towards studying and targeting breast cancer innervation.
PubMed: 38881512
DOI: 10.1111/jmi.13340 -
Biochemistry. Biokhimiia May 2024Induced pluripotent stem cells (iPSCs), capable of differentiating into any cell type, are a promising tool for solving the problem of donor organ shortage. In addition,... (Review)
Review
Induced pluripotent stem cells (iPSCs), capable of differentiating into any cell type, are a promising tool for solving the problem of donor organ shortage. In addition, reprogramming technology makes it possible to obtain a personalized, i.e., patient-specific, cell product transplantation of which should not cause problems related to histocompatibility of the transplanted tissues and organs. At the same time, inconsistent information about the main advantage of autologous iPSC-derivatives - lack of immunogenicity - still casts doubt on the possibility of using such cells beyond immunosuppressive therapy protocols. This review is devoted to immunogenic properties of the syngeneic and autologous iPSCs and their derivatives, as well as to the reasons for dysregulation of their immune tolerance.
Topics: Induced Pluripotent Stem Cells; Humans; Immune Tolerance; Cell Differentiation; Animals; Transplantation, Autologous
PubMed: 38880643
DOI: 10.1134/S0006297924050031 -
STAR Protocols Jun 2024Flow cytometry, single-cell RNA sequencing, and other analyses enable us to capture immune profiles of the tumor microenvironment. Here, we present a protocol to...
Flow cytometry, single-cell RNA sequencing, and other analyses enable us to capture immune profiles of the tumor microenvironment. Here, we present a protocol to characterize the immune profile of tumor-bearing mice. We describe steps for establishing mouse models and preparing single-cell suspensions from tumor tissue and other immune-related organs, which can be further analyzed by flow cytometry and other omics assays. We then detail procedures for staining, flow cytometry analysis, and phenotyping of the immune cell populations. For complete details on the use and execution of this protocol, please refer to Miyauchi et al..
PubMed: 38878286
DOI: 10.1016/j.xpro.2024.103139 -
Ultrasound in Medicine & Biology Jun 2024Both microbubble ultrasound contrast agents and acoustic phase change droplets (APCD) have been explored in hepatocellular carcinoma (HCC). This work aimed to evaluate...
OBJECTIVE
Both microbubble ultrasound contrast agents and acoustic phase change droplets (APCD) have been explored in hepatocellular carcinoma (HCC). This work aimed to evaluate changes to the HCC microenvironment following either microbubble or APCD destruction in a syngeneic pre-clinical model.
METHODS
Mouse RIL-175 HCC tumors were grown in the right flank of 64 immunocompetent mice. Pre-treatment, photoacoustic volumetric tumor oxygenation, and power Doppler measurements were obtained using a Vevo 3100 system (VisualSonics, Toronto, Canada). The experimental groups received a 0.1 mL bolus injection of either Definity ultrasound contrast agent (Lantheus Medical Imaging) or APCD fabricated by condensing Definity. Following injection, ultrasound destruction was performed using flash-replenishment sequences on a Sequoia with a 10L4 probe (Siemens) for the duration of enhancement. Tumor oxygenation and power Doppler measurements were then repeated immediately post-ultrasound treatment. Twenty-four hours post-treatment, animals were euthanized, and tumors were harvested and stained for CD31, Cleaved Caspase 3 and CD45.
RESULTS
Imaging biomarkers demonstrated a significant reduction in percent vascularity following either microbubble or APCD destruction in the tumor microenvironment ( p < 0.022) but no significant changes in tumor oxygenation (p = 0.12). Similarly, immunohistochemistry data demonstrated a significant decrease in CD31 expression (p < 0.042) and an increase in apoptosis (p < 0.014) in tumors treated with destroyed microbubbles or APCD relative to controls. Finally, a significant increase in CD45 expression was observed in tumors treated with APCD (p = 0.046), indicating an increase in tumor immune response.
CONCLUSION
Ultrasound-triggered destruction of both microbubbles and APCD reduces vascularity, increases apoptosis, and may also increase immune response in this HCC model.
PubMed: 38876912
DOI: 10.1016/j.ultrasmedbio.2024.05.015 -
Breast Cancer Research and Treatment Jun 2024Breast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs...
PURPOSE
Breast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action.
METHODS
The inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells.
RESULTS
UM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture.
CONCLUSION
These results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.
PubMed: 38874684
DOI: 10.1007/s10549-024-07372-0 -
Journal of Cancer Research and Clinical... Jun 2024With the development of immunotherapy research, the role of immune checkpoint blockade (ICB) in the treatment of cervical cancer has been emphasized, but many patients...
PURPOSE
With the development of immunotherapy research, the role of immune checkpoint blockade (ICB) in the treatment of cervical cancer has been emphasized, but many patients still can't receive long-term benefits from ICB. Poly ADP ribose polymerase inhibitor (PARPi) has been proved to exert significant antitumor effects in multiple solid tumors. Whether cervical cancer patients obtain better benefits from the treatment regimen of PARPi combined with ICB remains unclear.
METHODS
The alteration of PD-L1 expression induced by niraparib in cervical cancer cells and its underlying mechanism were assessed by western blot and immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR).The regulation of PTEN by KDM5A was confirmed using Chromatin immunoprecipitation (ChIP) assay and RNA interference. Analyzing the relationship between PD-L1 and immune effector molecules through searching online databases. Therapeutic efficacy of niraparib, PD-L1 blockade or combination was assessed in syngeneic tumor model. The changes of immune cells and cytokines in vivo was detected by immunohistochemistry (IHC) and qRT-PCR.
RESULTS
We found that niraparib upregulated PD-L1 expression and potentiated the antitumor effects of PD-L1 blockade in a murine cervical cancer model. Niraparib inhibited the Pten expression by increasing the abundance of KDM5A, which expanded PD-L1 abundance through activating the PI3K-AKT-S6K1 pathway. PD-L1 was positively correlated with immune effector molecules including TNF-α, IFN-γ, granzyme A and granzyme B based on biological information analysis. Niraparib increased the infiltration of CD8 T cells and the level of IFN-γ, granzyme B in vivo.
CONCLUSION
Our findings demonstrates the regulation of niraparib on local immune microenvironment of cervical cancer, and provides theoretical basis for supporting the combination of PARPi and PD-L1 blockade as a potential treatment for cervical cancer.
Topics: Uterine Cervical Neoplasms; Female; Humans; Animals; Piperidines; B7-H1 Antigen; Indazoles; Mice; Immune Checkpoint Inhibitors; Poly(ADP-ribose) Polymerase Inhibitors; Cell Line, Tumor
PubMed: 38869633
DOI: 10.1007/s00432-024-05819-x