Treatment Of Murine Model Research Articles

Remodeling the tumor immune microenvironment through hydrogel encapsulated G-Rh2 in situ vaccine and systemic immunotherapy

Ginsenoside Rh2 (G-Rh2) is a vital bioactive compound in Traditional Chinese Medicine, celebrated for its strong pharmacological properties, particularly its potent antitumor effects. However, its poor water solubility and limited bioavailability have necessitated the development of a novel drug delivery method. In this study, we utilized an indocyanine green carboxylic acid-hydroxypropyl cellulose-abietic acid-bovine serum albumin hydrogel (ICG-HPC-AA/BSA hydrogel) as a tumor in situ vaccine to enhance the permeability, retention, and tumor-targeted therapeutic efficacy of G-Rh2. We examined the therapeutic impact of a G-Rh2-loaded hydrogel combined with systemic PD-1 antibody treatment in murine models of H22 liver cancer and CT26 colon cancer. Additionally, we explored the immune microenvironment of the tumors influenced by this in situ vaccination strategy.

18F]AlF-CBP imaging of type I collagen for non-invasive monitoring of pulmonary fibrosis in preclinical models.

Pulmonary fibrosis is an irreversible scar-forming condition for which there is a lack of non-invasive and specific methods for monitoring its progression and therapy efficacy. However, the disease is known to be accompanied by collagen accumulation. Here, we developed a novel positron emission tomography (PET) probe targeting type I collagen to evaluate its utility for the non-invasive assessment of pulmonary fibrosis. We designed a 18F-labeled PET probe ([18F]AlF-CBP) to target type I collagen and evaluated its binding affinity, specificity and stability in vitro. PET with [18F]AlF-CBP, CT, histopathology, immunofluorescence, and biochemical indice were performed to assess and quantify type I collagen levels and pulmonary fibrosis progression and treatment in murine models. Dynamic PET/CT studies of [18F]AlF-CBP were conducted to assess lung fibrosis in non-human primate models. [18F]AlF-CBP was successfully prepared, and in vitro and in vivo tests showed high stability (> 95%) and type I collagen specificity (IC50 = 0.36 µM). The lungs of the fibrotic murine model showed more elevated probe uptake and retention compared to the control group, and there was a positive correlation between the radioactivity uptake signals and the degree of fibrosis (CT: R2 = 0.89, P < 0.0001; hydroxyproline levels: R2 = 0.89, P < 0.0001). PET signals also correlated well with mean lung density in non-human primate models of pulmonary fibrosis (R2 = 0.84, P < 0.0001). [18F]AlF-CBP PET imaging is a promising non-invasive method for specific monitoring of lung fibrosis progression and therapy efficacy.

Robust pancreatic tumor suppression by a novel combination treatment with anti-PD-1 immune checkpoint antibody and stroma modifying RNA oligonucleotide STNM01 in mice.

e14630 Background: Tumor stromal remodeling is an obstacle for immune checkpoint blockade therapies. STNM01 is a synthetic RNA oligonucleotide that represses carbohydrate sulfotransferase 15 (CHST15), which is responsible for tumor stromal remodeling. We have previously reported that STNM01 monotherapy by intratumoral injection repressed tumor stromal remodeling while increase tumor-infiltrating T lymphocytes (TILs) in pancreatic tumor-bearing mice. STNM01 was shown to increase TILs in patients with unresectable pancreatic cancer in a Phase I/IIa trial. In the present study, we investigated whether STNM01-mediated TIL enhancement improves anti-tumor effect of anti-PD-1 immune checkpoint antibody by combination treatment in murine models of pancreatic tumor. Methods: In mouse pancreatic cancer KPC and Pan02 subcutaneous syngeneic tumor models, mice were divided into 4 groups for treatment; 1) control, 2) STNM01 monotherapy, 3) anti-PD-1 antibody monotherapy, and 4) combination therapy with STNM01 and anti-PD-1 antibody. Intratumoral injections with 0.9-1.0 mg/mL of STNM01 or control and intraperitoneal administrations of anti-PD-1 antibody or control (5mg/kg) were then performed twice a week for 2 weeks. Mice were sacrificed after 2 week-treatment from baseline, and anti-tumor effects were evaluated by immunohistochemistry for KPC and flowcytometry for Pan02 model, respectively. Results: In the KPC-implanted syngeneic mouse model, combination treatment with intratumoral STNM01 and systemic anti-PD-1 antibody synergistically and robustly suppressed tumor growth as mean % tumor growth inhibition was over 80%. Tumor-infiltrating CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to anti-PD-1 monotherapy group. In the Pan02-implanted syngeneic mouse model, the combination treatment with STNM01 and anti-PD-1 significantly augmented necrosis area of tumor. Flowcytometry analyses showed that tumor-infiltrating CD3+, CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to control group. Although FoxP3+ Treg did not change, the ratio of CD8 to FoxP3 was significantly high only in the anti-PD-1 plus STNM01 combination therapy group. Notably, Ly6C+Ly6G+ granulocytic MDSCs dramatically diminished in the anti-PD-1 plus STNM01 combination therapy group, while anti-PD-1 monotherapy had no impact on tumoral MDSCs. Conclusions: The present work represents the first report of robust synergy between systemic anti-PD-1 antibody and single stroma modifying agent through intratumoral route of administration. Combination usage of STNM01 would provide a novel and clinically feasible therapeutic option to trigger remarkable effect of immune checkpoint inhibitors to this most hard-to-treat solid tumor.

First-in-Class HAT Activator (YF2) Combined with JAK/STAT Inhibitor (ruxolitinib) Unveils Potential Novel Treatment Approach for HDAC Inhibitor-Resistant CTCL

T-cell lymphomas are both aggressive and challenging to treat. This is in-part due to multiple epigenetic derangements found in T-cell lymphomas. One treatment strategy includes HDAC inhibitors; however, the duration of response is short for most patients which is consistent with many treatments for T-cell lymphoma. As a result, alternative treatments are required. YF2, a first-in-class histone acetyltransferase (HAT) activator, was invented at Columbia University. YF2 increases acetylation of histones by augmenting the activity of HAT enzymes. The JAK/STAT pathway contributes to CTCL lymphomagenesis through a number of cellular processes including cell cycle, proliferation, apoptosis, etc. JAKs activate STAT3, which activates the transcription of the anti-apoptotic Bcl-2-like protein, Bcl-XL. Additionally, HATs acetylate the pro-apoptotic p53 protein, increasing its stability and activity. Given the dependence on both the JAK/STAT pathway and epigenetic derangements in CTCL, we hypothesize that if the JAK inhibitor, ruxolitinib, induces apoptosis via modulation of Bcl-XL, and YF2 induces apoptosis via acetylation and activation of p53, then dual targeting of these complementary pathways could lead to synergistic apoptosis in CTCL. To study this hypothesis, we used two CTCL cell lines: HH [JAK wildtype (JAK WT)], and H9 [JAK1/3 gain-of-function mutated (JAK1/3 GOF)]. In addition, we generated a belinostat-resistant H9 cell line (H9-belino-R) by incrementally exposing H9 to increasing concentrations of belinostat. H9-belino-R resistance was observed at a 20-fold (IC 50 = 896.7nM, SEM ± 185.3) increase over that of the parental H9 (IC 50 = 43.8nM, SEM ± 4.61) ( figure 1). H9-belino-R retained significant resistance to other HDAC inhibitors such as romidepsin [(H9: IC 50 = 0.97nM (SEM ± 0.030), H9-belino-R: IC 50 = 1.38nM (SEM ± 0.028)] and panobinostat [H9: IC 50 = 3.11nM (SEM ± 0.19), H9-belino-R: IC 50 = 9.00nM (SEM ± 1.55)] as measured by the CellTiter-Glo Viability Assay. Interestingly, the IC 50 of the HAT activator, YF2, was retained between the resistant (14.84µM, SEM ± 0.46) and parental (12.45µM, SEM ± 0.27) cell lines ( figure 1). HH (JAK WT) showed significant cytotoxicity (63.5% live, 30.7% apoptotic, and 5.4% dead) after 4-day exposure to 10µM YF2 compared to limited cytotoxicity (93.3% live, 4.6% apoptotic, and 2.0% dead) in H9 (JAK GOF) as measured by flow cytometry with Annexin V. At baseline, active STAT3 (pSTAT3) was not detected in HH possibly contributing to its susceptibility to YF2 as the absence of pSTAT3 reduces the transcription Bcl-XL. As illustrated by figure 2, western blot analysis confirmed ruxolitinib-induced inhibition of active STAT3 (pSTAT3) with associated reduction in Bcl-XL expression in H9 and H9-belino-R. This may explain the observed synergistic cytotoxicity with YF2 and ruxolitinib in HH, H9 and H9-belino-R (Excess over Bliss scores: 4.85, 14.23, and 5.38 respectively) as determined by flow cytometry. In conclusion, the novel first-in-class HAT activator, YF2, retains activity in HDAC inhibitor-resistant CTCL. YF2's activity may be blunted by JAK1/3 GOF mutation; however, when combined with the JAK-inhibitor ruxolitinib, the response mirrored that of the HH (JAK WT) cell line. The combination of HAT activator and JAK inhibitor led to enhanced reduction of the anti-apoptotic protein Bcl-XL, potentially explaining synergistic apoptosis in CTCL. These preliminary findings provide us with evidence that suggests that the combination of YF2 and ruxolitinib can serve as a novel treatment combination for CTCL. Further study is planned to explore this treatment in murine models of disease.

Combining Treg Therapy With Donor Bone Marrow Transplantation: Experimental Progress and Clinical Perspective.

Donor-specific tolerance remains a goal in transplantation because it could improve graft survival and reduce morbidity. Cotransplantation of donor hematopoietic cells to achieve chimerism is a promising approach for tolerance induction, which was successfully tested in clinical trials. However, current protocols are associated with side effects related to the myelosuppressive recipient conditioning, which makes it difficult to introduce them as standard therapy. More recently, adoptive cell therapy with polyclonal or donor-specific regulatory T cells (Treg) proved safe and feasible in several transplant trials, but it is unclear whether it can induce tolerance on its own. The combination of both approaches-Treg therapy and hematopoietic cell transplantation-leads to chimerism and tolerance without myelosuppressive treatment in murine models. Treg therapy promotes engraftment of allogeneic hematopoietic cells, reducing conditioning requirements and enhancing regulatory mechanisms maintaining tolerance. This review discusses possible modes of action of transferred Treg in experimental chimerism models and describes translational efforts investigating the potent synergy of Treg and chimerism.

TIGIT-Fc Prolongs Corneal Allograft Survival in Mice by Upregulating TIGIT/CD226 Expression and the Proportion of Helios + Foxp3 + Treg Cells.

Reduction of graft rejection remains key issue for supporting long-term graft retention after corneal transplantation. The relevance of Treg in reduction of corneal allografts rejection has been demonstrated. It has been recently reported that in addition to Foxp3, Helios is also considered to be a marker of activated Treg. Helios + Foxp3 + Treg are considered to be the true immunosuppressive Treg. TIGIT is an immunosuppressive costimulatory molecule that was found to be highly expressed on the surface of Helios + Foxp3 + Treg. In this study, we aimed to explore whether supplementing TIGIT would result in an expansion and activation of Helios + Foxp3 + Treg thus to mediate an immune tolerance following corneal transplantation by administering topically and systemically TIGIT-Fc treatment in murine models. TIGIT-Fc treatment significantly improved the survival of corneal allograft compared with the control group. TIGIT-Fc treatment increased TIGIT/CD226 expression, the proportion of Helios + Foxp3 + Treg cells and an enhanced ex vivo suppressive effect from peripheral lymph nodes isolated Treg cells. Furthermore, the expression of Helios in corneal grafts was upregulated, whereas expression of CD226 and production of aqueous interferon-γ and VEGF were reduced by TIGIT-Fc treatment. TIGIT-Fc treatment could specifically upregulate Helios + Foxp3 + Treg-mediated immune response after allogeneic corneal transplantation via TIGIT/CD226-CD155 pathway which improves the survival of allografts.

Interleukin-27 Regulates the Function of the Gastrointestinal Epithelial Barrier in a Human Tissue-Derived Organoid Model.

Simple SummaryThe gut is lined by a single layer of epithelial cells, creating a barrier between the contents of the gut and the underlying tissue. This barrier also controls permeability (to determine if gut contents move to the tissue) and produces proteins that help with immune responses. These epithelial cells are damaged and do not function properly in inflammatory bowel diseases (IBD). IBD is common, and current treatments are not always effective and can have side effects. New treatments are needed. An alternative treatment target is the epithelial gut lining to promote healing. We used an experimental model of human gut tissue epithelial cells (called organoids) and aimed to define the role of the protein interleukin-27 (IL-27) in epithelial barrier function. We show that IL-27 restored permeability associated with changes in several proteins involved in this process, led to changes in genes involved in the immune response and reaction to bugs in the gut and led to enhanced healing and repair of an injury wound in the barrier. In summary, this study demonstrates a new function of IL-27 in the gut to aid epithelial barrier repair. This is a potential new treatment strategy for IBD.A treatment with direct healing effects on the gastrointestinal epithelial barrier is desirable for inflammatory bowel disease (IBD). Interleukin-27 (IL-27) is an immunoregulatory cytokine, and oral delivery is an effective treatment in murine models of IBD. We aimed to define IL-27 effects on the human gastrointestinal epithelial barrier. We characterised gene and protein expression of permeability mediators in a human colon-derived organoid model. Functional permeability was determined in an organoid-derived 2D monolayer by transepithelial electrical resistance. IL-27 effects on epithelial innate immune responses were assessed through expression of cytokines, anti-microbial peptides and MUC genes. IL-27 effects on wound healing and proliferation were determined in human colon epithelial cell lines. IL-27 led to restoration of permeability regulation following inflammatory cytokine insult (p = 0.001), associated with differential expression of tight junction mediators with decrease in claudin 2 (p = 0.024) and increase in claudin 4 (p < 0.001), E-cadherin (p < 0.001) and zona occludens (p = 0.0014). IL-27 evoked differential gene expression of epithelial-derived innate immune responses (reduced IL1B and IL18, and increased IL33, HBD1, MUC1 and MUC2; p < 0.012). IL-27 induced epithelial barrier wound healing through restitution (p < 0.001), and increased proliferation (p < 0.001) following injury. Overall, IL-27 provokes mucosal healing of the human gastrointestinal epithelial barrier.

Probable Mechanisms of Doxorubicin Antitumor Activity Enhancement by Ginsenoside Rh2.

Ginsenoside Rh2 increases the efficacy of doxorubicin (DOX) treatment in murine models of solid and ascites Ehrlich’s adenocarcinoma. In a solid tumor model (treatment commencing 7 days after inoculation), DOX + Rh2 co-treatment was significantly more efficacious than DOX alone. If treatment was started 24 h after inoculation, the inhibition of tumor growth of a solid tumor for the DOX + Rh2 co-treatment group was complete. Furthermore, survival in the ascites model was dramatically higher for the DOX + Rh2 co-treatment group than for DOX alone. Mechanisms underlying the combined DOX and Rh2 effects were studied in primary Ehrlich’s adenocarcinoma-derived cells and healthy mice’s splenocytes. Despite the previously established Rh2 pro-oxidant activity, DOX + Rh2 co-treatment revealed no increase in ROS compared to DOX treatment alone. However, DOX + Rh2 treatment was more effective in suppressing Ehrlich adenocarcinoma cell adhesion than either treatment alone. We hypothesize that the benefits of DOX + Rh2 combination treatment are due to the suppression of tumor cell attachment/invasion that might be effective in preventing metastatic spread of tumor cells. Ginsenoside Rh2 was found to be a modest activator in a Neh2-luc reporter assay, suggesting that Rh2 can activate the Nrf2-driven antioxidant program. Rh2-induced direct activation of Nrf2 might provide additional benefits by minimizing DOX toxicity towards non-cancerous cells.

Efficacy of Spironolactone Treatment in Murine Models of Cutaneous and Visceral Leishmaniasis.

Translational studies involving the reuse and association of drugs are approaches that can result in higher success rates in the discovery and development of drugs for serious public health problems, including leishmaniasis. If we consider the number of pathogenic species in relation to therapeutic options, this arsenal is still small, and each drug possesses a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. In the search for new drugs, we performed a drug screening of L. amazonensis promastigotes and intracellular amastigotes of fifty available drugs belonging to several classes according to their pharmacophoric group. Spironolactone, a potassium-sparing diuretic, proved to be the most promising drug candidate. After demonstrating the in vitro antileishmanial activity, we evaluated the efficacy on a murine experimental model with L. amazonensis and L. infantum. The treatment controlled the cutaneous lesion and reduced the parasite burden of L. amazonensis significantly, as effectively as meglumine antimoniate. The treatment of experimental visceral leishmaniasis was effective in reducing the parasite load on the main affected organs (spleen and liver) via high doses of spironolactone. The association between spironolactone and meglumine antimoniate promoted better control of the parasite load in the spleen and liver compared to the group treated with meglumine antimoniate alone. These results reveal a possible benefit of the concomitant use of spironolactone and meglumine antimoniate that should be studied more in depth for the future possibility of repositioning for leishmaniasis co-therapy.

Blockade of CD40L inhibits immunogenic maturation of lung dendritic cells: Implications for the role of lung iNKT cells in mouse models of asthma

Some studies have shown that maturation of dendritic cells (DCs) is modulated directly by pathogen components via pattern recognition receptors such as Toll-like receptors, but also by signal like CD40 ligand (CD40 L or CD154) mediated by activated T cells. Several reports indicate that invariant natural killer T (iNKT) cells up-regulate CD40 L upon stimulation and thereby induce activation and maturation of DCs through crosslink with CD40. Our previous findings indicated that iNKT cells promote Th2 cell responses through the induction of immunogenic maturation of lung DCs (LDCs) in the asthmatic murine, but its mechanism remains unclear. Therefore, we investigated the immunomodulatory effects of blockade of CD40 L using anti-CD40 L treatment on Th2 cell responses and immunogenic maturation of LDCs, and further analyzed whether these influences of blockade of CD40 L were related to lung iNKT cells using iNKT cell-deficient mice and the combination treatment of specific iNKT cell activation with anti-CD40 L treatment in murine models of asthma. Our findings showed that blockade of CD40 L using anti-CD40 L treatment attenuated Th2 cell responses in wild-type (WT) mice, but not in CD1d-deficient mice sensitized and challenged with ovalbumin (OVA) or house dust mite (HDM). Meanwhile, blockade of CD40 L down-regulated immunogenic maturation of LDCs in WT mice, but not in CD1d-deficient mice sensitized and challenged with OVA. Additionally, agonistic anti-CD40 treatment reversed the inhibitory effects of anti-CD40 L treatment on Th2 cell responses and LDC activation in an OVA-induced mouse model of asthma. Furthermore, LDCs from asthmatic mice treated with anti-CD40 L could significantly reduce the influence on Th2 cell responses in vivo and in vitro. Finally, α-Galactosylceramide plus anti-CD40 L treatment stimulated lung iNKT cells, but suppressed Th2 cell responses in the asthmatic mice. Taken together, our data raise an evidence that blockade of CD40 L attenuates Th2 cell responses through the inhibition of immunogenic maturation of LDCs, which may be at least partially related to lung iNKT cells in murine models of asthma.

Blockade of fibroblast activation protein in combination with radiation treatment in murine models of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic stroma with a poor lymphocyte infiltrate, in part driven by cancer-associated fibroblasts (CAFs). CAFs, which express fibroblast activation protein (FAP), contribute to immune escape via exclusion of anti-tumor CD8+ T cells from cancer cells, upregulation of immune checkpoint ligand expression, immunosuppressive cytokine production, and polarization of tumor infiltrating inflammatory cells. FAP is a post-proline peptidase selectively expressed during tissue remodeling and repair, such as with wound healing, and in the tumor microenvironment by cancer-associated fibroblasts. We targeted FAP function using a novel small molecule inhibitor, UAMC-1110, and mice with germline knockout of FAP and concomitant knock-in of E. coli beta-galactosidase. We depleted CAFs by adoptive transfer of anti-βgal T cells into the FAP knockout animals. Established syngeneic pancreatic tumors in immune competent mice were targeted with these 3 strategies, followed by focal radiotherapy to the tumor. FAP loss was associated with improved antigen-specific tumor T cell infiltrate and enhanced collagen deposition. However, FAP targeting alone or with tumor-directed radiation did not improve survival even when combined with anti-PD1 therapy. Targeting of CAFs alone or in combination with radiation did not improve survival. We conclude that targeting FAP and CAFs in combination with radiation is capable of enhancing anti-tumor T cell infiltrate and function, but does not result in sufficient tumor clearance to extend survival.

Allicin enhances antimicrobial activity of macrophages during Mycobacterium tuberculosis infection

Ethnopharmacological relevanceThe emergence of drug-resistant Mycobacterium tuberculosis (M.tb) strains has severely hampered global efforts towards tuberculosis (TB) eradication. The internationally accepted therapy “Directly Observed Treatment Short-course (DOTS)” is lengthy, and incorporates risks for the generation of drug-resistant M.tb variants. Multiple and extremely drug-resistant (MDR and XDR) variants of TB are now widespread throughout the globe, and totally drug-resistant (TDR) strains have appeared. Therefore, new classes of antibiotics are urgently needed to combat these deadly organisms. Historically, garlic is known to kill mycobacterial strains, and its active compound, allicin, kills various microorganisms. Here we have shown that allicin not only reduced the bacterial burden in the lungs of mice infected with Mycobacterium tuberculosis (M.tb), but also induces strong anti-tubercular immunity. Materials and methodsIn the present study, the anti-mycobacterial and immunomodulatory activity of garlic extract and its pure constituent allicin were demonstrated based on several in vitro and in vivo experiments in murine model of tuberculosis. Furthermore, the validation of study was done by immunoblots showing the modulation of MAPK and SAPK/JNK signaling by allicin in macrophages. ResultsHere, we report that allicin/garlic extract exhibits strong anti-mycobacterial responses in vitro and in vivo against drug-sensitive, MDR and XDR strains of TB. In addition to direct killing, allicin also induced pro-inflammatory cytokines in macrophages. Moreover, allicin/garlic extract treatment in murine models of infection resulted in induction of strong protective Th1 response, leading to drastic reduction in mycobacterial burden. These results indicated that allicin/garlic extract has both antibacterial and immunomodulatory activity. Furthermore, garlic extract reversed the immune dampening effects of frontline anti-TB drugs. ConclusionAllicin/garlic extract alone or as an adjunct to classical antibiotics holds great promise for treatment of drug-sensitive as well as drug-resistant TB. These results warrant further study and validation of allicin for treatment of TB.

Toll-like receptor agonist rMBP-NAP enhances antitumor cytokines production and CTL activity of peripheral blood mononuclear cells from patients with lung cancer.

Toll-like receptor (TLR) agonists are known for their ability to inhibit tumor progression via enhancing antitumor cytokines production and cytotoxic T lymphocyte (CTL) activity. Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP) has been reported as a novel TLR agonist for antitumor treatment in murine models. The present study aimed to determine the potential and efficacy of the rMBP-NAP for antitumor treatment prior to further clinical trials. The rMBP-NAP was expressed and purified for subsequent experiments. Peripheral blood mononuclear cells (PBMCs) from health donors and patients with lung cancer (LC) were incubated with PBS and 0.2 mg/ml rMBP-NAP. Antitumor cytokines production was assayed using ELISA and reverse transcription-quantitative polymerase chain reaction analysis. The cytolytic activity of PBMCs and the number of Interferon-γ (IFN-γ)-secreting cells were assayed using lactate dehydrogenase and Enzyme-linked ImmunoSpot assays, respectively. The results from the present study revealed that the expression of IFN-γ, interleukin (IL)-2, tumor necrosis factor-α and IL-12 of PBMCs from patients with LC and healthy donors were significantly increased following treatment with rMBP-NAP (P<0.05). Additionally, rMBP-NAP significantly upregulated the number of IFN-γ-secreting cells in PBMCs and prominently increased the cytotoxic activity of PBMCs (P<0.05). Furthermore, the expression of TLR2 was significantly enhanced following rMBP-NAP stimulation (P<0.05), which indicated that rMBP-NAP may serve an antitumor role via TLR2 signaling pathways. Overall, these results demonstrated that rMBP-NAP possesses the potential to be a novel immunomodulatory candidate drug and requires further evaluation in clinical trials.

Calprotectin in rheumatic diseases.

Calprotectin is a heterodimer formed by two proteins, S100A8 and S100A9, which are mainly produced by activated monocytes and neutrophils in the circulation and in inflamed tissues. The implication of calprotectin in the inflammatory process has already been demonstrated, but its role in the pathogenesis, diagnosis, and monitoring of rheumatic diseases has gained great attention in recent years. Calprotectin, being stable at room temperature, is a candidate biomarker for the follow-up of disease activity in many autoimmune disorders, where it can predict response to treatment or disease relapse. There is evidence that a number of immunomodulators, including TNF-α inhibitors, may reduce calprotectin expression. S100A8 and S100A9 have a potential role as a target of treatment in murine models of autoimmune disorders, since the direct or indirect blockade of these proteins results in amelioration of the disease process. In this review, we will go over the biologic functions of calprotectin which might be involved in the etiology of rheumatic disorders. We will also report evidence of its potential use as a disease biomarker. Impact statement Calprotectin is an acute-phase protein produced by monocytes and neutrophils in the circulation and inflamed tissues. Calprotectin seems to be more sensitive than CRP, being able to detect minimal residual inflammation and is a candidate biomarker in inflammatory diseases. High serum levels are associated with some severe manifestations of rheumatic diseases, such as glomerulonephritis and lung fibrosis. Calprotectin levels in other fluids, such as saliva and synovial fluid, might be helpful in the diagnosis of rheumatic diseases. Of interest is also the potential role of calprotectin as a target of treatment.

Use of non-invasive imaging to monitor response to aflibercept treatment in murine models of colorectal cancer liver metastases.

The liver is the most frequent metastatic site in colorectal cancer (CRC), and relevant orthotopic in vivo models are needed to study the efficacy of anticancer drugs in the metastatic setting. A challenge when utilizing such models is monitoring tumor growth during the experiments. In this study, experimental liver metastases were established in nude mice by splenic injection of the CRC cell lines HT29 and HCT116, and the mice were treated with the antiangiogenic drug aflibercept. Tumor growth was monitored using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Aflibercept treatment was well tolerated and resulted in increased animal survival in HCT116, but not in HT29, while inhibited tumor growth was observed in both models. Treatment efficacy was monitored with high precision using MRI, while BLI detected small-volume disease with high sensitivity, but was less accurate in end-stage disease. Apparent diffusion coefficient (ADC) values obtained by diffusion weighted MRI (DW-MRI) were highly predictive of treatment response, with increased ADC corresponding well with areas of necrosis observed by histological evaluation of aflibercept-treated xenografts. The results showed that the efficacy of the antiangiogenic drug aflibercept varied between the two models, possibly reflecting unique growth patterns in the liver that may be representative of human disease. Non-invasive imaging, especially MRI and DW-MRI, can be used to effectively monitor tumor growth and treatment response in orthotopic liver metastasis models.

Abstract 3207: Influence of tumor mutation burden on response to anti-PD-1 treatment in murine models of melanoma

Abstract Therapeutic antibodies directed against immune checkpoints, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1), have recently shown remarkable clinical benefits, particularly in metastatic melanoma and non-small cell lung cancer (NSCLC), cancers largely caused by chronic exposure to mutagenic agents. Recent studies have focused on identifying genomic and immune predictors of response, but consistent, robust biomarkers have yet to be fully characterized. However, some data suggests a high non-synonymous tumour mutation burden is associated with clinical benefit in melanoma patients who received anti-CTLA-4 therapy. A similar correlation was also found in NSCLC patients treated with anti-PD-1. Using mouse models of BRAFV600E-driven melanoma, we examined the relationship between tumour mutation burden and response to the immune checkpoint inhibitor anti-PD-1. Chronic exposure to ultraviolet radiation (UVR) was used to increase the number of tumour non-synonymous single nucleotide variants (SNVs), and thus mutational load. Responses to PD-1 blockade were compared between tumours having low (∼10 SNVs) and high (&amp;gt;100 SNVs) mutation burden and tumours were subsequently subjected to comprehensive molecular and immune profiling using whole exome sequencing, immunohistochemistry, and flow cytometry. Our initial data suggest there is no significant correlation between tumour mutation burden and response in our model of BRAFV600E-driven melanoma, and we will present a detailed analysis of low- and high-mutation burden tumours in light of their response to PD-1 blockade. Consistent with previous studies, our results highlight that integrated molecular characterization of tumour tissue and associated microenvironment in larger cohorts is needed to identify robust determinants of response to immune checkpoint inhibitors. Citation Format: Elena Galvani, Kate Hogan, Gabriela Gremel, Amaya Viros, Amit K. Mandal, Matthew Smith, Jacqueline Swan, Antonia Banyard, Garry Ashton, Nathalie Dhomen, Richard Marais. Influence of tumor mutation burden on response to anti-PD-1 treatment in murine models of melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3207.

Abstract B179: Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers

Abstract Different components of the tumor microenvironment hamper the immune response against cancer cells and are a critical impediment for the success of cancer immunotherapy. Among these, the accumulation of regulatory T cells (Treg) and the reduction of the effector T cells (Teff)/Treg ratio favors tumor progression and is associated with worse prognosis in several human cancers. Treg elimination in the tumor microenvironment can be achieved through antibodies that target molecules expressed preferentially in Treg and that induce antibody-mediated cell cytotoxicity (ADCC). CTLA-4 is highly expressed on tumor-infiltrating Treg and therapy with anti-CTLA-4 has been effective for cancer treatment in murine models and in humans. In mice, we and others have shown that anti-CTLA-4 depletes Treg in the tumor but not in peripheral lymphoid organs. This site specificity in mice is given by the expression of the Fcγ-receptor IV (FcγRIV) in myeloid cells present in the tumor microenvironment but not in the periphery. However, the role of FcγRs in the effectiveness of anti-CTLA-4 therapy or therapies with other immunomodulatory antibodies in humans is not known. We therefore investigated the density of targets of clinically relevant immunomodulatory antibodies in different subpopulations of tumor-infiltrating immune cells as well as the expression of FcγR subtypes in murine tumor models. Because of the inter-species differences in the repertoire and the cellular distribution of FcγRs, we also studied this in humanized-FcγR (huFcγR) mice, in which there is no expression of murine FcγRs but of their human homologues. We compared this with samples of human melanoma and observed surprisingly similar expression profiles of both FcγRs and targets of immunomodulatory antibodies, including CTLA-4. We then asked whether anti-CTLA-4 would also lead to selective Treg depletion in the tumor microenvironment in huFcγR mice. Using an antibody targeting mCTLA-4 with a human IgG1 isotype, we observed that was the case. Furthermore, mutation of the Fc region of the antibody to enhance its affinity to activating huFcγRs resulted in more effective selective Treg depletion in the tumor. These findings are not only relevant for CTLA-4 but also for other targets expressed preferentially on Treg instead of Teff and must be taken into account when designing immunomodulatory antibodies for clinical use. Citation Format: Frederick Arce Vargas, Andrew J. Furness, Marta H. Lesko, Martin Pule, Jeffrey V. Ravetch, Karl S. Peggs, Sergio A. Quezada. Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B179.

Abstract 400: MEK inhibition enhances immune checkpoint blockade treatment of murine models of neuroblastoma

Abstract Background: The tumor microenvironment (TME) plays an important role in neuroblastoma (NBL) biology. Infiltrations of tumor-associated macrophages with alterations in patterns of pro-inflammatory genes are associated with poor prognosis in NBL. However, the role of regulatory T-cells in NBL remains unknown. As several genes in MAPK pathway are among recurrent mutations in neuroblastomas, and the production of pro-inflammatory mediators in immune cells is MAPK-dependent, we hypothesized targeted kinase inhibitor in combination with check-point blockade could exert synergistic effects on the TME's response to tumor cells. Therefore, we assessed the efficacy of combined therapy using a transgenic MYCN non-amplified neuroblastoma murine model driven by SV40 large T antigen (NB-Tag). Methods: Three human neuroblastoma cell lines and two NB-Tag derived mouse cell-lines were used for in vitro cell proliferation assay. For in vivo tumor growth models, combinations of cyclophosphamide, topotecan, trametinib, anti-CTLA4, and anti-PD1 therapies were studied in NB-Tag transgenic and transplantable subcutaneous (NB-SQ) murine models. Results: In vitro studies demonstrated trametinib had the highest anti-proliferative activity compared to other kinase inhibitors, and it effectively blocked cell cycle arrest in G1 phase. These anti-proliferative effects could not be rescued by co-culturing tumor cells with murine or human macrophages. In NB-Tag mice, which develop neuroblastoma spontaneously at 12 weeks, daily oral administration of trametinib (0.6mg/Kg) at pre-tumoral age (10 wk) significantly impaired tumor growth by 17 weeks (1424 mm3 in controls vs. 43 mm3 in treated mice). Treatment of 15 week-old NB-Tag mice (visible tumor by MRI) with trametinib after chemotherapy administration (5-days of Cyclophosphamide + Topotecan) also significantly impaired tumor regrowth (volume four weeks post-chemo, 491 vs. 42 mm3, p = 0.037), and more importantly, treatment increased the survival of NB-Tag mice compared to control (median survival: control = 24.5 wk, treated = 35 wk, p&amp;lt;0.0001). Trametinib treatment also showed similar results in the NB-SQ model. Addition of immune checkpoint blockade of NB-SQ mice with anti-CTLA4 and anti-PD1 to treatment with trametinib further increased survival compared to controls (p&amp;lt;0.0001). Tumors and macrophages collected from mice treated with trametinib showed effective block in phosphorylation of ERK. Transgenic animals that grew despite trametinib treatment showed strong STAT3 phosphorylation suggesting this pathway as an escape mechanism. Conclusions: Our results provide strong evidence that MEK inhibition combined with checkpoint blockade significantly inhibited tumor formation in a syngeneic subcutaneous model. These findings indicate opportunities to enhance antitumor immunity with the potential to produce durable clinical responses in children with neuroblastomas. Citation Format: Sakunthala Muthugounder, Long Hung, Randall Chan, Jin Kim, Soheila Shirinbak, Hiroyuki Shimada, Shahab Asgharzadeh. MEK inhibition enhances immune checkpoint blockade treatment of murine models of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2015-400

Modulation of Interaction of Human Osteoprogenitor Cells with Hematopoietic Stem and Progenitor Cells

Osteoprogenitor cells (OPCs) are marrow microenvironmental cells known to modulate hematopoietic stem and progenitor cells (HSPCs). Specifically, OPCs regulate HSPCs in response to Parathyroid hormone (PTH) treatment in murine models. However, the role of OPCs in human HSPC regulation and whether human OPCs can be manipulated is poorly understood. Niche stimulation is an appealing strategy to aid in the treatment of hematopoietic dysfunction. Myelodysplastic syndromes (MDS) are clonal disorders with ineffective hematopoiesis resulting in cytopenias and risk of transformation to acute leukemia (AML). In mouse models, disruption of the osteolineage cells can contribute to initiation of ineffective hematopoiesis with phenotypic features of MDS. Our long term goal is to utilize microenvironmental stimulation as a therapeutic tool to improve hematopoietic disorders. We hypothesized that human cells isolated from the marrow fraction containing spicules harbor HSPC supportive cells, which can be manipulated to improve HSPC support. Moreover we hypothesized that OPC number and function is impaired by dysplasia-initiated microenvironmental disruption as a potential mechanism for reduced support of HSPCs and ineffective hematopoiesis. Our objective was to isolate human bone marrow spicule associated cells (SACs) and define their ability to support HSPCs, determine the impact of PTH treatment of SAC/HSPCs interactions and characterize dysplasia-induced osteolineage changes in human MDS and AML bone marrow. To achieve this objective, we used normal as well as MDS/AML patient-derived OPCs using a mouse-human co-culture system.Human bone marrow SACs isolated by collagenase digestion were either used for co-culture, analyzed with flow cytometry or cultured in mineralization media in limited dilutions.To assess the potential impact of PTH on human OPC interaction with HSPCs, we developed a 7 day co-culture of human bone marrow SACs treated with either vehicle or PTH, with mouse Lineage- Sca1+ c-Kit+ (LSK) hematopoietic progenitor cells. At the end of the co-culture, all cells present were used for competitive transplantation. Transplant experiments demonstrated that PTH treatment of the human bone marrow SACs leads to improved function of the co-cultured LSK cells as demonstrated by significantly improved engraftment of the LSK cells after transplant into irradiated C57/bl6 recipient mice when sampled at pre-specified time points over a 20-week period (N=12, 2-way ANOVA; p < 0.05).Flow cytometry analysis showed that mature (Lin- CD31- CD146+ CD105-) and immature osteolineage (Lin- CD31- CD146+ CD105+) cells were present in SACs and more abundant compared to within BMMCs (1% vs 0.1% and 0.24% vs 0.12% for the same patient). Notably, the putative HSC-supportive MSC pool was increased in SACs vs BMMCs (0.052% vs 0.019%). The presence of OPCs was functionally confirmed using colony forming unit osteoblasts (CFU-OBs). CFU-OB frequency was calculated using L-Calc TM (StemCell technologies). Among normal donors the frequency of CFU-OBs was low in marrow donors >50 years old compared to <50 years old donors (2.240e-005 ± 3.300e-006 N=2 vs. 0.0001146 ± 4.163e-005 N=9). We identified non-statistically significant decrease in the frequency of CFU-OBs in bone marrow SACs from MDS patients compared to normal donors (1.090e-005 ± 1.400e-006 N=2 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.179); and similar decrease in frequency of osteoprogenitor cells in the bone marrow aspirates from AML patients compared to normal donors (2.303e-005 ± 9.371e-006 N=3 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.251). These data support our hypothesis that OPCs in patients with MDS and AML are negatively impacted compared to normal bone marrow.These data demonstrate that human SACs contain HSPC-supportive cells which can be stimulated to improve HSPC function. Human SACs comprise MSCs and osteolineage cells including osteoprogenitor cells. Aging decreases OPC pools in SACs. Our data in our small sample also suggest that dysplastic bone marrow microenvironment may negatively impact OPCs, which may in turn decrease OPC support of HPSCs. PTH treatment in our in-vitro model shows the potential to improve the interaction between the OPCs and HSPCs, resulting in amelioration of HSC function. Together these data suggest a strategy where targeting the MDS microenvironment may add to the currently available treatment modalities. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Abstract 1248: Intralesional injection with PV-10 induces a systemic anti-tumor immune response in murine models of breast cancer and melanoma.

Abstract PV-10 is a 10% solution of Rose Bengal that is currently being examined as a novel cancer therapeutic. In melanoma patients, intralesional injection (IL) of PV-10 has led to regression of injected lesions as well as distant metastases. In this study, we examined the efficacy and potential immune mechanism of PV-10 treatment in murine models of breast cancer and melanoma. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions (p&amp;lt;0.05 compared to IL-PBS-treated mice). A significant increase in survival was observed in mice treated with PV-10. To examine immune response, MT901-specific IFN-gamma production and cytotoxicity were measured in splenocytes collected from mice treated with IL-PBS or IL-PV-10. MT901-bearing mice treated with IL-PV-10 demonstrated enhanced IFN-gamma production (992 ± 453 pg/ml) compared to splenocytes from PBS-treated mice (174 ± 105, p&amp;lt;0.05). In addition, a significant increase in lysis of MT-901 cells by T cells after PV-10 treatment was observed (p&amp;lt;0.01 compared to PBS-treated mice). No lysis of irrelevant CT-26 cells was detected. In a murine model of melanoma, B16-F10 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL-PV-10 led to regression of the injected lesion as well as distant B16 melanoma lung metastases. In B16-bearing mice, treatment with IL-PV-10 led to the induction of T cells that produced IFN-gamma in response to B16 tumors but not irrelevant tumor (p&amp;lt;0.05) and demonstrated specific lysis of B16 (p&amp;lt;0.01 compared to T cells isolated from PBS-treated mice). In total, these studies support the induction of tumor-specific T cell-mediated immunity after single treatment with IL-PV-10 in multiple histologic subtypes. The immune mechanism of PV-10 ablation in cutaneous melanoma patients is currently under investigation at our institution. Citation Format: Shari A. Pilon-Thomas, Amy Weber, Krithika Kodumudi, Lisa Kuhn, Paul Toomey, Amod Sarnaik. Intralesional injection with PV-10 induces a systemic anti-tumor immune response in murine models of breast cancer and melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1248. doi:10.1158/1538-7445.AM2013-1248