Renca Tumors Research Articles

Silver nanoparticle induced immunogenic cell death can improve immunotherapy

Cancer immunotherapy is often hindered by an immunosuppressive tumor microenvironment (TME). Various strategies are being evaluated to shift the TME from an immunologically ‘cold’ to ‘hot’ tumor and hereby improve current immune checkpoint blockades (ICB). One particular hot topic is the use of combination therapies. Here, we set out to screen a variety of metallic nanoparticles and explored their in vitro toxicity against a series of tumor and non-tumor cell lines. For silver nanoparticles, we also explored the effects of core size and surface chemistry on cytotoxicity. Ag-citrate-5 nm nanoparticles were found to induce high cytotoxicity in Renca cells through excessive generation of reactive oxygen species (ROS) and significantly increased cytokine production. The induced toxicity resulted in a shift of the immunogenic cell death (ICD) marker calreticulin to the cell surface in vitro and in vivo. Subcutaneous Renca tumors were treated with anti-PD1 or in combination with Ag-citrate-5 nm. The combination group resulted in significant reduction in tumor size, increased necrosis, and immune cell infiltration at the tumor site. Inhibition of cytotoxic CD8 + T cells confirmed the involvement of these cells in the observed therapeutic effects. Our results suggest that Ag-citrate-5 nm is able to promote immune cell influx and increase tumor responsiveness to ICB therapies.

Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors

BackgroundTargeting kinases presents a potential strategy for treating solid tumors; however, the therapeutic potential of vaccines targeting kinases remains uncertain.MethodsAdenovirus (Ad) vaccines encoding Aurora kinase A (AURKA) or cyclin-dependent kinase...

Time-course RNAseq data of murine AB1 mesothelioma and Renca renal cancer following immune checkpoint therapy

Time-critical transcriptional events in the immune microenvironment are important for response to immune checkpoint blockade (ICB), yet these events are difficult to characterise and remain incompletely understood. Here, we present whole tumor RNA sequencing data in the context of treatment with ICB in murine models of AB1 mesothelioma and Renca renal cell cancer. We sequenced 144 bulk RNAseq samples from these two cancer types across 4 time points prior and after treatment with ICB. We also performed single-cell sequencing on 12 samples of AB1 and Renca tumors an hour before ICB administration. Our samples were equally distributed between responders and non-responders to treatment. Additionally, we sequenced AB1-HA mesothelioma tumors treated with two sample dissociation protocols to assess the impact of these protocols on the quality transcriptional information in our samples. These datasets provide time-course information to transcriptionally characterize the ICB response and provide detailed information at the single-cell level of the early tumor microenvironment prior to ICB therapy.

Enhanced Theranostic Efficacy of 89Zr and 177Lu-Labeled Aflibercept in Renal Cancer: A Viable Option for Clinical Practice.

Vascular endothelial growth factor (VEGF) targeted therapy serves as an important therapeutic approach for renal cancer, but its clinical effectiveness is unsatisfactory. Moreover, there is a lack of reliable biomarkers for preoperative assessment of tumor VEGF expression. This study aimed to explore the potential for further applications of 177Lu/89Zr-labeled aflibercept (Abe), a VEGF-binding agent, in imaging visualization of VEGF expression and therapy for renal cancer. To determine specificity uptake in renal cancer, BALB/c mice with VEGF-expressing Renca tumor were intravenously injected with [89Zr]Zr-Abe, [177Lu]Lu-Abe, or Cy5.5-Abe and the blocking group was designed as a control group. PET, SPECT, and fluorescence images were acquired, and the biodistribution of [89Zr]Zr-Abe and [177Lu]Lu-Abe was performed. Additionally, the [177Lu]Lu-Abe, [177Lu]Lu-Abe-block, 177Lu only, Abe only, and PBS groups were compared for evaluation of the therapeutic effect. To assess the safety, we monitored and evaluated the body weight, blood biochemistry analysis, and whole blood analysis and major organs were stained with hematoxylin and eosin after [177Lu]Lu-Abe treatment. DOTA-Abe was successfully labeled with 177Lu and Df-Abe with 89Zr in our study. The uptake in tumor of [89Zr]Zr-Abe was significantly higher than that of [89Zr]Zr-Abe-block (P < 0.05) and provided excellent tumor contrast in PET images. [177Lu]Lu-Abe demonstrated promising tumor-specific targeting capability with a high and persistent tumor uptake. The standardized tumor volume of [177Lu]Lu-Abe was significantly smaller than those of other treatment groups (P < 0.05). [177Lu]Lu-Abe also had smaller tumor volumes and reduced expression of VEGF and CD31 compared to those of the control groups. Fluorescence images demonstrate higher tumor uptake in the Cy5.5-Abe group compared to the Cy5.5-Abe-block group (P < 0.05). In conclusion, [89Zr]Zr-Abe enables noninvasive analysis of VEGF expression, serving as a valuable tool for assessing the VEGF-targeted therapy effect. Additionally, all of the findings support the enhanced therapeutic efficacy and safety of [177Lu]Lu-Abe, making it a viable option for clinical practice in renal cancer.

Targeting oxidative phosphorylation to increase the efficacy of immune-combination therapy in renal cell carcinoma

BackgroundImmune checkpoint inhibitors (ICIs) are the standard of care for metastatic renal cell carcinoma (RCC); however, most patients develop de novo or acquired resistance to ICIs. Oxidative phosphorylation (OXPHOS) has...

Development of a New Focal Mouse Model of Bone Metastasis in Renal Cell Carcinoma.

Developing animal models of bone metastasis in renal cell carcinoma (RCC) is challenging as immunodeficient mice are required. The aim of this study was to develop a simple immune model of RCC bone metastasis. RENCA tumor cells were injected into the right femurs of BALB/c mice. Sixty mice were grouped into each twenty-mouse group according to the tumor cell concentration, and the presence or absence and extent of bone metastasis in the total length of the femur were compared using hematoxylin and eosin staining of the excised tissues. Bone metastasis was significantly higher in the high concentration group than in the other groups (p<0.05), with 10 mice developing bone metastasis at two weeks and nine mice developing bone metastasis at three weeks. The extent of bone metastasis was significantly greater in the high concentration group than in the other groups (p<0.05). Multiple logistic regression analysis was performed to examine the factors influencing bone metastasis, and only the high concentration was a significant factor (p<0.05). We developed a normal immunity mouse model of local bone metastasis from RCC. This model could prove valuable for research into the treatment of bone metastases in RCC.

Cancer Immunotherapy with Lipid Nanoparticles Loaded with a Stimulator of Interferon Genes Agonist against Renal Tumor Lung Metastasis.

Metastatic renal cell carcinoma (RCC) has a poor prognosis, and the major organ of metastasis is the lung. Immunotherapy with immune checkpoint inhibitors (ICIs) is the first-line therapy, but the response rates are low. Thus, the development of a more effective immunotherapy against metastatic RCC would be highly desirable. We previously demonstrated how a stimulator of an interferon gene (STING) agonist-loaded lipid nanoparticles (STING-LNPs) significantly activates natural killer (NK) cells and induces an antitumor effect against cases of melanoma lung metastasis that have shown ICI resistance. In this study, we evaluated the potential of using STING-LNPs in the treatment of lung metastatic RCC (Renca). An intravenous injection of STING-LNPs drastically decreased the amount of Renca tumor colonies. In contrast, monotherapies using ICIs showed no antitumor effect, and even a combination of ICI and STING-LNP therapies failed to enhance the antitumor effects. The main effector cells would be NK cells, and the activation of NK cells by the STING-LNPs may avoid the increased expression of immune checkpoint molecules. These findings provide useful insights into the development of an effective immunotherapy against metastatic RCC.

SGN-B7H4V, an investigational vedotin ADC directed to the immune checkpoint ligand B7-H4, shows promising activity in preclinical models

BackgroundSGN-B7H4V is a novel investigational vedotin antibody–drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline...

A modified IL-18 drug in combination with CTLA-4 blockade enhances anti-tumor efficacy in preclinical models of renal cell carcinoma

Abstract Background Cytokine-based drugs are currently being explored as alternative cancer immunotherapies. While the cytokine interleukin-18 (IL-18) has immunostimulatory effects, it is negatively regulated by a secreted high-affinity binding protein, IL-18BP, that functions as an immune checkpoint that limits IL-18’s efficacy as a cancer therapeutic. A modified version of IL-18, termed “decoy-resistant” or DR-18, that can avoid trapping by IL-18BP while still maintaining its immune signaling potential, has recently been developed. DR-18 has shown promising preclinical activity in melanoma and colorectal murine models, including potential synergy with anti-PD-1 therapy, and is currently in Phase I trials. In this study, we aim to test the efficacy and determine the cellular mechanism of action of DR-18 in combination with immune checkpoint inhibitors (ICIs) in immunocompetent preclinical models of renal cell carcinoma (RCC), with the goal of establishing the basis for testing these combinations in early phase clinical trials. Methods We engrafted tumors subcutaneously using two different syngeneic, immunocompetent murine RCC models: Renca and RAG. Mice were treated with single-agent DR-18 and combinations of DR-18 with single- and dual-agent anti-PD-1 and anti-CTLA-4. Tumor growth and survival were monitored. In the Renca model, plasma was collected at early time-points and cytokine/chemokine levels were profiled using a 31-plex discovery assay. Single-cell RNA and TCR sequencing was also performed on Renca tumors. Additionally, immune cell depletion studies were conducted in the Renca model with antibodies targeting CD8, CD4, NK cells, and interferon-gamma. Results In the Renca model, DR-18 monotherapy modestly inhibited tumor growth and prolonged survival. The effects were comparable to single- and dual-agent ICIs. Adding PD-1 blockade to DR-18 did not enhance efficacy whereas the addition of anti-CTLA-4 to DR-18 significantly increased anti-tumor effects. Triple-therapy (DR-18 plus anti-PD-1 plus anti-CTLA-4) did not further inhibit tumor growth or prolong survival compared to the doublet (DR-18 plus anti-CTLA-4). The RAG model was more sensitive to ICIs but produced similar results, again showing modest anti-tumor activity of single-agent DR-18 and enhanced benefit of combining with anti-CTLA-4 but not anti-PD-1. Cytokine/chemokine profiling revealed significantly elevated levels of IP-10 (CXCL10) and MIG (CXCL9) after one cycle of DR-18 plus anti-CTLA-4 compared to control and single-agent treatments, suggesting that these chemokines may be key early mediators of the anti-tumor immune response. Single-cell transcriptomic analysis demonstrated changes in intra-tumoral T cell, macrophage, and granulocyte populations with DR-18 plus anti-CTLA-4 relative to other regimens, including enrichment of CD8+ precursor and terminally exhausted T cells and a neutrophil population associated with interferon signaling. Additionally, single-cell TCR analysis showed a reduction in intra-tumoral clonotype diversity with DR-18 plus anti-CTLA-4 compared to other treatments. Immune cell depletion studies identified CD8+ T cells, NK cells, and interferon-gamma, but not CD4+ T cells, as equally required for efficacy of DR-18 plus anti-CTLA-4. Conclusions In this study, we identify DR-18, an IL-18-based drug engineered with resistance to a secreted decoy-receptor protein, in combination with anti-CTLA-4 as having enhanced anti-tumor activity in preclinical models of RCC. This regimen was associated with a more pro-inflammatory immune microenvironment. Further investigation is ongoing to elucidate the cellular mechanism of action of this regimen more fully and lay the groundwork for clinical testing of DR-18-based combination therapy in RCC. In the future, testing novel partner agents outside of anti-PD-1/CTLA-4 and using RCC models of ICI-resistance could be particularly informative and clinically relevant.

SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma.

Immune checkpoint blockade (ICB) demonstrates durable clinical benefits in a minority of patients with renal cell carcinoma (RCC). We aimed to identify the molecular features that determine the response and develop approaches to enhance it. We investigated the effects of SET domain-containing protein 2 (SETD2) loss on the DNA damage response pathway, the cytosolic DNA-sensing pathway, the tumor immune microenvironment, and the response to ataxia telangiectasia and rad3-related (ATR) and checkpoint inhibition in RCC. ATR inhibition activated the cyclic GMP-AMP synthase (cGAS)-interferon regulatory factor 3 (IRF3)-dependent cytosolic DNA-sensing pathway, resulting in the concurrent expression of inflammatory cytokines and immune checkpoints. Among the common RCC genotypes, SETD2 loss is associated with preferential ATR activation and sensitizes cells to ATR inhibition. SETD2 knockdown promoted the cytosolic DNA-sensing pathway in response to ATR inhibition. Treatment with the ATR inhibitor VE822 concurrently upregulated immune cell infiltration and immune checkpoint expression in Setd2 knockdown Renca tumors, providing a rationale for ATR inhibition plus ICB combination therapy. Setd2-deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or combination therapy with VE822 than Setd2-proficient tumors. Moreover, SETD2 mutations were associated with a higher response rate and prolonged overall survival in patients with ICB-treated RCC but not in patients with non-ICB-treated RCC. SETD2 loss and ATR inhibition synergize to promote cGAS signaling and enhance immune cell infiltration, providing a mechanistic rationale for the combination of ATR and checkpoint inhibition in patients with RCC with SETD2 mutations.

Assessments of therapeutic effects according to timings for combined therapy with axitinib and immune check point inhibitor in a mouse renal cell carcinoma model

Recently, several types of systemic therapy using tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI) have been performed for advanced renal cell carcinoma (aRCC) patients; however, the optimal strategy of sequential treatment with these agents has not been well established. The objective of this study was to determine the differences of therapeutic effects according to timing for the introduction of TKI and ICI using a mouse RCC, RenCa model. The effects of combined treatment of TKI and/or ICI with axitinib, anti-mouse programmed death (PD)-1, or PD-ligand 1 (PD-L1) antibody on tumor growth and survival after subcutaneous and intravenous injection of RenCa cells, respectively, were compared according to three different treatment schedules: simultaneous administration, initial axitinib administration, and initial ICI administration. Infiltrating patterns of lymphocytes into tumors after combined treatments were evaluated by immunohistochemical staining. In mice treated with anti-PD-1 and anti-PD-L1 antibodies, significantly marked inhibitory effects on subcutaneous growth of tumors were observed in the simultaneous and initial ICI treatment groups, but not the group with the initial axitinib administration, compared to controls without treatment. Survival intervals of mice after intravenous injection of RenCa cells were significantly longer in the simultaneous and initial ICI administration, but not the initial axitinib administration, compared to the control. Furthermore, both CD8+ to CD3+ and CD8+ to CD11b+ T-lymphocyte ratios in subcutaneous RenCa tumors were significantly higher in the simultaneous and initial ICI administration, but not the initial axitinib administration, compared to the control. Favorable control against aRCC progression may be achieved by administering TKI and ICI simultaneously or ICI followed by TKI.

A cisplatin conjugate with tumor cell specificity exhibits antitumor effects in renal cancer models

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer and is notorious for its resistance to both chemotherapy and small-molecule inhibitor targeted therapies. Subcellular targeted cancer therapy may thwart the resistance to produce a substantial effect.MethodsWe tested whether the resistance can be circumvented by subcellular targeted cancer therapy with DZ-CIS, which is a chemical conjugate of the tumor-cell specific heptamethine carbocyanine dye (HMCD) with cisplatin (CIS), a chemotherapeutic drug with limited use in ccRCC treatment because of frequent renal toxicity.ResultsDZ-CIS displayed cytocidal effects on Caki-1, 786-O, ACHN, and SN12C human ccRCC cell lines and mouse Renca cells in a dose-dependent manner and inhibited ACHN and Renca tumor formation in experimental mouse models. Noticeably, in tumor-bearing mice, repeated DZ-CIS use did not cause renal toxicity, in contrast to the CIS-treated control animals. In ccRCC tumors, DZ-CIS treatment inhibited proliferation markers but induced cell death marker levels. In addition, DZ-CIS at half maximal inhibitory concentration (IC50) sensitized Caki-1 cells to small-molecule mTOR inhibitors. Mechanistically, DZ-CIS selectively accumulated in ccRCC cells’ subcellular organelles, where it damages the structure and function of mitochondria, leading to cytochrome C release, caspase activation, and apoptotic cancer cell death.ConclusionsResults from this study strongly suggest DZ-CIS be tested as a safe and effective subcellular targeted cancer therapy.

Stimulation of antitumor immunity by FoxP3-targeting PROTAC

CD4 + regulatory T cells (Tregs) play a central role in regulating and suppressing anti-tumor immune responses. FoxP3 is a transcription factor and master regulator of the Treg lineage. We developed and characterized a proteolysis targeting chimeric (PROTAC) drug that targets FoxP3 (PF). PF was created by linking the FoxP3 binding peptide P60 to pomalidomide, a ligand for E3 ligase. Ternary complex formation between PF, FoxP3, and cereblon (component of an E3 ligase) was confirmed using surface plasmon resonance assay (cooperativity factor of 2.27). PF decreased mouse and human FoxP3 expression in vitro in a proteasome-dependent manner. In mice, PF decreased FoxP3 in both the spleen and peripheral lymphocytes. PF-treated lymphocytes (human or mice) were better at stimulating CD8 + lymphocyte proliferation and activation. PF treatment decreased RENCA tumor growth in mice. PF enhanced antitumor immunity associated with αPD1 or mTOR inhibitor (mTORi). Lymphocytes from mice treated with PF and mTORi showed reduced metastatic tumor growth in untreated mice, providing further evidence for an adaptive immune response as the mechanism of action. We showed that PF binds FoxP3 and decreases FoxP3 expression in Tregs, reducing Treg function and generating antitumor immunity.

Abstract 6789: The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models

Abstract Background: TGFB1 mediated immune resistance is one of the major mechanisms of immune suppression utilized across multiple tumor types. Immune resistance imparted by TGFB1 is mediated through its pleiotropic effects on vasculature, fibrogenesis and regulatory/effector immune cells within the tumor microenvironment. Blockade of TGFB1 (TGFBi) will likely improve response to immunotherapy. IL-12 is a cytokine that through IFNg induction, promotes type 1 inflammatory response, M1 macrophages and effector CD8 T cell response. Combining TGFB1 blockade with IL-12 may maximize therapeutic benefits through simultaneously reducing immunosuppression and enhancing anti-tumor immune response. Current studies have developed a vaccinia-based immunotherapy, combining enhanced systemic virus delivery to CXCR3 ligand rich tumors and locally expressed IL-12 and TGFBi within the tumor microenvironment, for efficient control of multiple tumor models. Methods: An oncolytic vaccinia virus expressing CXCR3, IL-12 and a TGFB1 antagonizing mini-monomer was constructed (VET3-TGI) and the expression and function of the transgenes were confirmed. Using in vivo mouse RENCA, EMT-6 and MC38 tumor models, the functionality and therapeutic efficacy of VET-TGI were tested with comparison to control virus. Post-mortem analysis was used to analyze the impact of VET3-TGI on immune/stromal/endothelial milieu of the tumors and to determine toxicity profile. Results: VET3-TGI infected cells expressed CXCR3 and showed enhanced migration to CXCR3 ligands in vitro and improved systemic delivery to tumors expressing CXCR3 ligands in vivo, even in the face of pre-existing anti-viral immunity. IL-12 expression and TGFBi blockade of TGFB1 mediated suppression of CD8 T cell proliferation were confirmed in vitro. In vivo mouse studies using EMT6, RENCA and MC38 tumor models demonstrated potent therapeutic activity, including 100% CRs, even at doses several logs below equivalent clinical doses and in multiple models. Mechanism of activity studies suggested that the therapeutic efficacy of VET3-TGI is associated with considerable modification of the tumor microenvironment. In addition, preliminary toxicity studies demonstrated the safety of VET3-TGI in mouse models. Conclusions: VET3-TGI demonstrated an ability to reduce immunosuppression and dramatically enhance antitumor immune response leading to safe and potent therapeutic activity in multiple mouse tumor models. This data led to the selection of VET3-TGI as our lead clinical candidate. A human version of the virus is currently undergoing clinical manufacture and toxicology testing. Citation Format: Ravikumar Muthuswamy, Steve Thorne. The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6789.

Abstract 6257: Developing HDAC inhibitors as immunomodulators

Abstract Immune checkpoint inhibitors (ICIs) are standard of care for several solid tumor types, including renal cell carcinoma (RCC). However, the clinical success of these therapies is dampened by the fact that the majority of patients demonstrate either inherent resistance to treatment or develop secondary resistance following an initial response. One key mechanism of tumor-driven ICI resistance is the generation and recruitment of suppressive immune cell types, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which coordinate to suppress anti-tumor T cell responses. Thus, there is a critical need for the development of novel combination strategies aimed at mitigating the presence and function of suppressive immune cells to improve the efficacy of ICIs in the clinic. Our laboratory has previously demonstrated that histone deacetylase inhibition (HDACi) is associated with a decrease in both the number and function of tumor-infiltrating Tregs and MDSCs in the RENCA tumor model. Thus, the overall goal of these studies both preclinically and clinically is to investigate the impact of the immunomodulatory effects of HDACi in combination with ICI therapy, with the overall hypothesis that HDACi will improve the efficacy of anti-PD1 therapy in RCC. Our pre-clinical data support this hypothesis by demonstrating increased tumor control and survival in RENCA tumor-bearing mice when treated with a combination of anti-PD1 and HDACi compared to single agent therapy. These increases were also associated with an increase in the activation state of anti-tumor immune cells, as demonstrated by increased surface expression of MHC Class II molecules on macrophages and a decrease in phenotypically defined monocytic MDSCs. ATACseq and RNAseq data generated from combination-treated animals also outline a gene expression signature associated with both myeloid and T cell activation. Interestingly, our clinical studies also afforded us the opportunity to investigate potential biomarkers associated with responder status, which will allow us to better predict patient outcomes and administer therapies accordingly. Accumulating evidence from two independent clinical trials run by our group suggests that high levels of suppressive CD11b+HLA-DRlo MDSCs at baseline in the peripheral blood is associated with lack of clinical response to the combination of HDACi and ICI. Overall, these data support the rationale for the combination of ICI and HDACi in the clinic, and provide a potentially useful biomarker through which we can predict the success of combination therapy. Citation Format: Sean Colligan, Li Shen, Christopher Rupert, Jonathan Bard, Roberto Pili. Developing HDAC inhibitors as immunomodulators [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 6257.

Tumoral Endoglin (CD105) Regulates the Tumor Microenvironment, Tumorigenicity and Therapeutic Response in Renal Cell Carcinoma

<h3>Purpose/Objective(s)</h3> Endoglin (CD105) is a transmembrane glycoprotein that serves as a co-receptor for the TGF-β signaling cascade. Due to its elevated expression on tumor associated vasculature of highly vascular cancers, it is categorized as a tumor associated antigen (TAA). Although CD105 has been largely studied as an endothelial cell marker and mediator of angiogenesis in various cancers including Renal cell carcinoma (RCC), the effect of its expression by tumor cells has not been fully elucidated in immune-competent models. Here we address the implication of tumoral CD105 on the tumor microenvironment (TME) and response to a <i>Listeria-monocytogenes</i> based immunotherapeutic vaccine directed against CD105 based on the hypothesis that "<i>Tumoral CD105 contributes to the aggressive phenotype of RCC and further drives response to immunotherapy"</i>. <h3>Materials/Methods</h3> A mutant version of Renca that is deficient in CD105(CD105KO-Renca) was generated from murine Renca cell line using CRISPR/cas9. In an immunocompetent model syngeneic to male BALB/C mice, subcutaneously implanted control Renca (Scr-Renca) or CD105 KO-Renca tumors were monitored for tumor growth. Tumor tissues from either group were subjected to flow cytometry and gene expression using RT-qPCR. The contribution of tumoral CD105 to neovascularization was determined by matrigel plug angiogenesis assay followed by hemoglobin quantification. Lastly, we elucidated how tumoral expression of CD105 affects the response to a <i>Listeria-</i>based vaccine directed against CD105 (Lm-LLO-CD105) in a subcutaneous immunocompetent mouse model. <h3>Results</h3> CD105KO-Renca displays increased potential for early tumor formation, but the tumor progression is reduced compared to the Scr-Renca. Deletion of tumoral CD105 impacted the TME by increasing the population of multicytokine (IFNγ⁺ IL-2⁺TNFα⁺) producing CD8⁺ and CD4⁺ T cells and reducing infiltration of myeloid derived suppressor cells (MDSCs) and M2-macrophages. Further, tumoral CD105 contributes to angiogenesis as observed by a reduction in the hemoglobin content and CD31mRNA and VEGFa mRNA in Renca-CD105KO tumor tissues. Finally, Lm-LLO-CD105 was able to control tumor growth in the Scr-Renca tumor bearing mice but not in the CD105KO-Renca tumor bearing mice. Lm-LLO-CD105 increases the CD8⁺ T cell infiltration, production of cytokines and reduces the population of CD4⁺Foxp3⁺Tregs in the TME of Scr-Renca but not in the CD105-KO-Renca tumors. <h3>Conclusion</h3> Tumoral CD105 serves as an independent prognostic that is important for both tumor progression and modulation of the RCC's immunological phenotype. Furthermore, CD105 deficient RCC did not respond to an immunotherapeutic strategy that is dependent on CD8⁺ T cell recruitment. Altogether, our data suggests that tumoral CD105 is an important mediator of tumor progression and response to immunotherapy.

Demonstrating Tumor Vascular Disrupting Activity of the Small-Molecule Dihydronaphthalene Tubulin-Binding Agent OXi6196 as a Potential Therapeutic for Cancer Treatment.

Simple SummaryVascular-disrupting agents promise significant therapeutic efficacy against solid tumors by selectively damaging tumor-associated vasculature. Following administration of the phosphate prodrug OXi6197, dynamic bioluminescence imaging revealed dose-responsive vascular shutdown in MDA-MB-231 human breast tumor xenografts and for the first time murine RENCA kidney tumors. Rapid vascular disruption was observed, which continued over 24 h. Histology showed vascular congestion, hemorrhage and necrosis. Twice-weekly doses of OXi6197 caused a significant growth delay in MDA-MB-231 breast tumors and many RENCA kidney tumors growing orthotopically in mice.The vascular disrupting activity of a promising tubulin-binding agent (OXi6196) was demonstrated in mice in MDA-MB-231 human breast tumor xenografts growing orthotopically in mammary fat pad and syngeneic RENCA kidney tumors growing orthotopically in the kidney. To enhance water solubility, OXi6196, was derivatized as its corresponding phosphate prodrug salt OXi6197, facilitating effective delivery. OXi6197 is stable in water, but rapidly releases OXi6196 in the presence of alkaline phosphatase. At low nanomolar concentrations OXi6196 caused G2/M cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells and monolayers of rapidly growing HUVECs underwent concentration-dependent changes in their morphology. Loss of the microtubule structure and increased bundling of filamentous actin into stress fibers followed by cell collapse, rounding and blebbing was observed. OXi6196 (100 nM) disrupted capillary-like endothelial networks pre-established with HUVECs on Matrigel®. When prodrug OXi6197 was administered to mice bearing orthotopic MDA-MB-231-luc tumors, dynamic bioluminescence imaging (BLI) revealed dose-dependent vascular shutdown with >80% signal loss within 2 h at doses ≥30 mg/kg and >90% shutdown after 6 h for doses ≥35 mg/kg, which remained depressed by at least 70% after 24 h. Twice weekly treatment with prodrug OXi6197 (20 mg/kg) caused a significant tumor growth delay, but no overall survival benefit. Similar efficacy was observed for the first time in orthotopic RENCA-luc tumors, which showed massive hemorrhage and necrosis after 24 h. Twice weekly dosing with prodrug OXi6197 (35 mg/kg) caused tumor growth delay in most orthotopic RENCA tumors. Immunohistochemistry revealed extensive necrosis, though with surviving peripheral tissues. These results demonstrate effective vascular disruption at doses comparable to the most effective vascular-disrupting agents (VDAs) suggesting opportunities for further development.

Abstract 4200: Characterization of acquired resistant models to therapies targeting the PD-1/PD-L1 axis demonstrates model-dependent mechanisms

Abstract The underlying mechanisms of acquired resistance to the immune checkpoint inhibitory (ICI) PD-1 and PD-L1 antibodies remain largely unknown and need to be further explored. In this study, we established and analyzed robust ICI resistance in in vivo models in order to identify new mechanisms and genes involved in acquired resistance. Resistant in vivo models were obtained by serial treatment/reimplantation cycles in immunocompetent mice bearing MC38, MB49, MBT2, TyrNRas or RENCA tumors. Spectral cytometry was applied to characterize modifications in the tumor immune microenvironment. RNAseq analyses were performed to identify differences in gene expression profiles between sensitive parental models compared to their resistant counterparts. Alterations in the tumor immune microenvironment were highly heterogeneous amongst resistant models, and are thus considered representative for the diversity expected in patients. Each resistant model displayed multiple modifications in the tumor immune infiltrate in comparison to the respective wild type model, involving selected lymphoid and/or myeloid subpopulations. For instance, in anti-PD-1 and anti-PD-L1 resistant MC38 models, we observed a remarkable increase of FoxP3+CD4+ T cells while CD8+ Tem cells were decreased in anti PD-1 resistant models. We also found that the type 2 macrophage content was increased in the anti PD-1 resistant MB49 model. Gene profiling analyses demonstrated the variability associated with acquisition of a resistance phenotype. Alterations of pathways previously reported to be associated with resistance to ICIs were observed in some of our models while other genes appeared to be highly de-regulated in several models. For instance, in the anti PD-L1 resistant MBT2 model many signaling pathways were found to be up-regulated, such as HIF1α, TGFβ, and ERK/MAPK signaling. Several therapeutic combinations, in particular targeting alternative immune checkpoints, were found to reverse the resistance phenotype in selected models. Moreover, in accordance with flow cytometry results, in the anti PD-1 resistant MC38 model we observed a significant delay in tumor growth when a TNFR1 antibody was combined with a PD-1 antibody (p&amp;lt;0.001). Acquired in vivo resistant models displayed strong diversity, both in terms of alterations of the tumor immune microenvironment and tumor gene expression profiles. Our model library, which may be enriched in the future with several other variants developed using the same methodology, provides an innovative tool to better understand the complexity and diversity of resistance to ICI and test resistance reversal strategies. Citation Format: Morgane Denis, Chloé Grasselly, Pierre-Antoine Choffour, Anne Wierinckx, Doriane Mathé, Kamel Chettab, Anne Tourette, Nolan Talhi, Fabian Birzele, Elsa Kress, Lars Petter Jordheim, Christian Klein, Eva-Laure Matera, Charles Dumontet. Characterization of acquired resistant models to therapies targeting the PD-1/PD-L1 axis demonstrates model-dependent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4200.

Abstract 6206: Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging

Abstract Tumor growth requires a functioning vascular network and offers an attractive therapeutic target. The invasive proliferating neovasculature of tumors lacks pericyte support and exhibits increased permeability thereby offering a unique, potentially selective target for anticancer therapy. Most vascular disrupting agents (VDAs) inhibit microtubule formation, causing rapid morphological changes in endothelia cells resulting in dramatically increased vessel permeability, cellular detachment, vessel occlusion, and vessel wall damage [doi: 10.3390/molecules26092551]. Highly vascular kidney tumors are expected to be particularly sensitive to VDAs. We are evaluating OXi8007, a water soluble indole phosphate pro-drug derivative of the first generation VDA combretastatin. The prodrug OXi8007 is stable in saline but releases active OXi8006 is the presence of alkaline phosphate. Preliminary pharmacokinetic data benchmarked using 13C-labeled OXi8006 and OXi8007 show rapid vascular clearance and conversion to the glucuronide conjugate in vivo. Doses up to 700 mg/kg were tolerated by mice. Luciferase-expressing RENCA tumor cells were implanted orthotopically in the right kidney capsule of syngeneic BALB/c mice. Bioluminescence Imaging (BLI) was applied weekly to assess tumor growth and acute response to OXi8007. Following addition of luciferin substrate subcutaneously BLI signal from the tumor reached a maximum within 10 minutes. When BLI was repeated 4 hrs after 250 or 350 mg/kg OXi8007 IP, the generated signal was &amp;lt;1% of baseline signal indicating acute vascular disruption. Multispectral Optoacoustic Tomography (MSOT) revealed matching acute changes in tumor vascular oxygenation and additionally showed distinct heterogeneity. An oxygen gas breathing challenge indicated lower oxygen saturation in the tumor following treatment. Histology showed massive hemorrhage following both doses. BLI signal recovered after 1 to 2 days and repeated administration of OXi8007 again caused acute vascular shutdown. These results match previous observations in breast tumor models [doi: 10.1016/j.canlet.2015.08.021] indicating opportunities for therapeutic application. Citation Format: Hashini I. Wanniarachchi, Regan Schuetze, Lorena Arango, Khagendra Hamal, Graham J. Carlson, Cyprian Pavlich, Yuling Deng, Mary L. Trawick, Kevin G. Pinney, Li Liu, Ralph P. Mason. Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6206.

PD59-12 MICROWAVE ABLATION COMBINED WITH ANTI-PD-1/CTLA-4 THERAPY INDUCES ANTI-TUMOR IMMUNE RESPONSE TO RENAL CELL CARCINOMA IN A MURINE MODEL

You have accessJournal of UrologyCME1 May 2022PD59-12 MICROWAVE ABLATION COMBINED WITH ANTI-PD-1/CTLA-4 THERAPY INDUCES ANTI-TUMOR IMMUNE RESPONSE TO RENAL CELL CARCINOMA IN A MURINE MODEL Run-Qi Guo, Jin-Zhao Peng, and Xiao-Guang Li Run-Qi GuoRun-Qi Guo More articles by this author , Jin-Zhao PengJin-Zhao Peng More articles by this author , and Xiao-Guang LiXiao-Guang Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002644.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Our current study was designed to investigate the characteristics of immune responses induced by MWA and dual immune checkpoint inhibitors (ICIs) in tumor tissues from tumor-bearing mice, with purpose of providing new insight into immune suppressive mechanisms limiting MWA efficacy and exploring the potential of dual ICIs in combination with MWA in cancer therapy. METHODS: First, untreated tumor assessment was performed to determine whether untreated tumor growth was suppressed. Mice with tumors established on the left kidney and right flank were randomly divided into 4 groups: the control group, the MWA group, the ICI group, and the MWA+ ICI group. Then, a rechallenge test was performed to determine whether systemic anti-tumor immunity was established. RESULTS: Although the volume of the untreated tumors continued to increase, tumor growth rates in the ICI group and the MWA+ICI group were significantly lower. CD8+ T cells were only significantly higher in the MWA+ICI group, while Treg cells were significantly lower in the MWA group, the ICI group and the MWA+ICI group. The MWA+ICI group also showed the highest amount of IFN-γ. Histopathologic studies revealed that cytotoxic T lymphocyte-associated antigen 4 expression (CTLA-4) in the distant tumor was significantly lower in the ICI group and the MWA+ICI group. MWA treatment led to an increase in programmed death ligand 1 (PD-L1)/ programmed death receptor 1 (PD-1) expression; however, after combining with ICI treatment, the expression decreased. In the tumor rechallenge test, tumor rejection was apparent in 16.7% of the mice in the MWA group, 50% of the mice in the ICI group, and 66.7% of the mice in the MWA+ICI group, whereas no mice in the control group showed tumor rejection. CONCLUSIONS: This study demonstrated that MWA induced systemic anti-tumor immunity was enhanced by combined use of anti PD-1/CTLA-4 therapy in a murine RENCA tumor model. The mice that received the combination therapy showed suppression of untreated tumor growth and tumor rejection. This treatment regimen is a potential clinical approach to enhance systemic anti-tumor immunity in patients with metastatic renal cell carcinoma. Source of Funding: This study was supported by Beijing Hospital Scientific Research Starting Foundation for PhD/MD under Grant BJ-2019-135 to Dr. Guo © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e1023 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Run-Qi Guo More articles by this author Jin-Zhao Peng More articles by this author Xiao-Guang Li More articles by this author Expand All Advertisement PDF DownloadLoading ...