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Supplementary Data from Therapeutic Potential of Adult Bone Marrow–Derived Mesenchymal Stem Cells in Prostate Cancer Bone Metastasis

Purpose: Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow–derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor κB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer.Experimental Design: The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro–computed tomography, immunohistochemistry, and histomorphometry.Results: Data indicated significant (P < 0.001) inhibition of tumor growth and restoration of bone in mice treated with unmodified and modified mesenchymal stem cells. Detailed analysis suggested that the donor mesenchymal stem cell inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis.Conclusions: Overcoming the limitation of the number of mesenchymal stem cells available in the bone can provide significant amelioration for osteolytic damage without further modification. (Clin Cancer Res 2009;15(23):7175–85)

PDF file - 358K, MDSC(+bone mets), which become osteoclasts was the only population that had elevated NO levels

PDF file - 100K, MDSC(+bone mets) did not express macrophage marker F4/80 during osteoclast differentiation and can induce bone destruction in vivo

PDF file - 372K, MDSC(+bone mets) was the only population that differentiated into osteoclasts

PDF file - 205K, Western blot analysis of HIF-1α, ERK and PI3K in MDSC from different tissue sources

PDF file - 381K, The phenotype of MDSC in the lung and spleen from lung cancer challenged mice (S1); Reduced tumor burden in mice from Gem and SODmim+Gem therapy groups (S2); Combination therapy reduced infiltration of neutrophils but not macrophages in tumor (S3); Combination therapy was efficient in reducing tumor growth in catalase deficient mice by decreasing MDSC infiltration and improving the memory CD8+ T cell response (S4); Persistent memory CD8+ T cell subsets expand following re-encounter with i.v. challenged LLC cells (S5); Adoptively transferred memory cells persist and reduce MDSC infiltration and tumor burden in LLC-rechallenged recipient mice (S6); Adoptively transferred memory cells expand upon encounter with tumor cells (S7).

PDF file - 103K, Nitric oxide levels are elevated in MDSC(+bone mets) as they differentiated into osteoclasts

PDF file - 169K, MDSC(+bone mets) from gemcitabine treated mice failed to differentiate into osteoclasts

PDF file - 108K, In vivo bone destruction mediated by MDSC requires NO production

PDF file - 143K, Isolation and characterization of MDSC from murine breast cancer model

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PDF file - 18K, Antibodies used for characterization of Immune Cell Phenotypes.

Purpose and Appropriate Sample TypesThis panel was designed to characterize the immune cell landscape in the mouse tumor micro-environment as well as mouse lymphoid tissues (e.g., spleen). Previous Optimized Multicolor Immunofluorescence Panels (OMIP) with conventional cytometry were examples of high-quality fluorescent-based flow cytometry panels to characterize either the T-cell compartment or the myeloid compartment [1, 2]. The advent of spectral cytometry has enabled sufficient markers to be included in a panel to comprehensively characterize the immune cell landscape including both the T-cell and the myeloid cell compartments. In this body of work, we demonstrated that we could measure the frequency and characterize the functional status of CD4 T cells, CD8 T cells, regulatory T cells, NK cells, B cells, macrophages, granulocytes, monocytes, & dendritic cells. This panel is especially useful for understanding the immune landscape in “cold” preclinical tumor models with very low immune cell infiltration, and for investigating how therapeutic treatments may modulate the immune landscape.

BackgroundProgrammed cell death protein 1 (PD-1) and CTLA4 combination blockade enhances clinical efficacy in melanoma compared with targeting either checkpoint alone; however, clinical response improvement is coupled with increased risk of developing immune-related adverse events (irAE). Delineating the mechanisms of checkpoint blockade-mediated irAE has been hampered by the lack of animal models that replicate these clinical events.MethodsWe have developed a mouse model of checkpoint blockade-mediated enterocolitis via prolonged administration of an Fc-competent anti-CTLA4 antibody.ResultsSustained treatment with Fc-effector, but not Fc-mutant or Fc-null, anti-CTLA4 antagonist for 7 weeks resulted in enterocolitis. Moreover, combining Fc-null or Fc-mutant CTLA4 antagonists with PD-1 blockade results in potent antitumor combination efficacy indicating that Fc-effector function is not required for combination benefit.ConclusionThese data suggest that using CTLA4 antagonists with no Fc-effector function can mitigate gut inflammation associated with anti-CTLA4 antibody therapy yet retain potent antitumor activity in combination with PD-1 blockade.

Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.

// Cara C. Schafer 1 , Yong Wang 1 , Kenneth P. Hough 1 , Anandi Sawant 2 , Stefan C. Grant 1 , Victor J. Thannickal 1 , Jaroslaw Zmijewski 1 , Selvarangan Ponnazhagan 2 , Jessy S. Deshane 1 1 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 2 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA Correspondence to: Jessy S. Deshane, email: treena@uab.edu Keywords: indoleamine 2,3-dioxygenase, myeloid-derived suppressor cells, lung cancer, metabolism, combination therapy Received: March 01, 2016 Accepted: September 13, 2016 Published: September 26, 2016 ABSTRACT Indoleamine 2,3-dioxygenase (IDO) has been implicated in immune evasion by tumors. Upregulation of this tryptophan (Trp)-catabolizing enzyme, in tumor cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME), leads to Trp depletion that impairs cytotoxic T cell responses and survival; however, exact mechanisms remain incompletely understood. We previously reported that a combination therapy of gemcitabine and a superoxide dismutase mimetic promotes anti-tumor immunity in a mouse model of lung cancer by inhibiting MDSCs, enhancing polyfunctional response of CD8 + memory T cells, and extending survival. Here, we show that combination therapy targets IDO signaling, specifically in MDSCs, tumor cells, and CD8 + T cells infiltrating the TME. Deficiency of IDO caused significant reduction in tumor burden, tumor-infiltrating MDSCs, GM-CSF, MDSC survival and infiltration of programmed death receptor-1 (PD-1)-expressing CD8 + T cells compared to controls. IDO -/- MDSCs downregulated nutrient-sensing AMP-activated protein kinase (AMPK) activity, but IDO -/- CD8 + T cells showed AMPK activation associated with enhanced effector function. Our studies provide proof-of-concept for the efficacy of this combination therapy in inhibiting IDO and T cell exhaustion in a syngeneic model of lung cancer and provide mechanistic insights for IDO-dependent metabolic reprogramming of MDSCs that reduces T cell exhaustion and regulates anti-tumor immunity.

Background A growing body of evidence indicates a positive correlation between expression of human antimicrobial peptide leucin leucin 37 (LL-37) and progression of epithelial cancers, including prostate cancer (PCa). Although the molecular mechanisms for this correlation has not yet been elucidated, the primary function of LL-37 as a chemotactic molecule for innate immune effector cells suggests its possible association in coordinating protumorigenic mechanisms, mediated by tumor-infiltrating immune cells. Methods To investigate protumorigenic role(s) of cathelicidin-related antimicrobial peptide (CRAMP), a murine orthologue of LL-37, the present study compared tumor growth kinetics between mouse PCa cell lines with and without CRAMP expression (TRAMP-C1 and TRAMP-C1CRAMP-sh, respectively) in immunocompetent mice. CRAMP-mediated chemotaxis of different innate immune cell types to the tumor microenvironment (TME) was observed in vivo and confirmed by in vitro chemotaxis assay. The role of CRAMP in differentiation and polarization of immature myeloid progenitors (IMPs) to protumorigenic type 2 macrophages (M2) in TME was determined by adoptive transfer of IMPs into mice bearing CRAMP(+) and CRAMP(−) tumors. To differentiate protumorigenic events mediated by tumor-derived CRAMP from host immune cell-derived CRAMP, tumor challenge study was performed in CRAMP-deficient mice. To identify mechanisms of CRAMP function, macrophage colony stimulating factor (M-CSF) and monocyte chemoattractant protein 1 (MCP-1) gene expression was analyzed by QRT-PCR and STAT3 signaling was determined by immunoblotting. Results Significantly delayed tumor growth was observed in wild-type (WT) mice implanted with TRAMP-C1CRAMP-sh cells compared to mice implanted with TRAMP-C1 cells. CRAMP(+) TME induced increased number of IMP differentiation into protumorigenic M2 macrophages compared to CRAMP(−) TME, indicating tumor-derived CRAMP facilitates differentiation and polarization of IMPs toward M2. Tumor challenge study in CRAMP deficient mice showed comparable tumor growth kinetics with WT mice, suggesting tumor-derived CRAMP plays a crucial role in PCa progression. In vitro study demonstrated that overexpressed M-CSF and MCP-1 in TRAMP-C1 cells through CRAMP-mediated autocrine signaling, involving p65, regulates IMP-to-M2 differentiation/polarization through STAT3 activation. Conclusion Altogether, the present study suggests that overexpressed CRAMP in prostate tumor initially chemoattracts IMPs to TME and mediates differentiation and polarization of early myeloid progenitors into protumorigenic M2 macrophages during PCa progression. Thus, selective downregulation of CRAMP in tumor cells in situ may benefit overcoming immunosuppressive mechanisms in PCa. Prostate © 2016 Wiley Periodicals, Inc.

Undesired immune responses against protein therapeutics may adversely affect the pharmacokinetics, efficacy, and safety of the product. The presence of anti-drug-antibodies (ADA) has been the key determinant of immunogenic responses. Here we describe the use of a capillary electrophoresis platform for the identification of ADAs against several experimental camelid VHH biologics (Nanobodies®). Hereafter, we refer to this assay as WESADA. We modified the Wes platform by ProteinSimple to screen serum samples for ADA against covalently linked multi-modular Nanobodies and compared it to standard ADA methodologies. We were able to identify ADA positive samples and determine which individual VHH module in a multivalent Nanobody construct stimulated the predominant ADA response. WESADA requires denaturation of the experimental immobilized drug, which could affect recognition of the immunogenic epitope and alter ADA signal. To address this issue, we demonstrated that signal can be immunodepleted by pre-incubation of serum samples with native Nanobody. This capillary electrophoresis based approach allows for rapid analysis without the need for individually tailored assay optimization or reagent labeling, while consuming small amounts of sample and drug. It also allows for the simultaneous ADA analysis of multiple targets of different molecular size in the same experimental sample. WESADA is not intended to replace traditional ADA assay formats, but it facilitates the expedient immunogenic assessment of a large number of experimental drug candidates in the early developmental space.

Bone fractures heal with overlapping phases of inflammation, cell proliferation, and bone remodeling. Osteogenesis and angiogenesis work in concert to control many stages of this process, and when one is impaired it leads to failure of bone healing, termed a nonunion. During fracture repair, there is an infiltration of immune cells at the fracture site that not only mediate the inflammatory responses, but we hypothesize they also exert influence on neovasculature. Thus, further understanding the effects of immune cell participation throughout fracture healing will reveal additional knowledge as to why some fractures heal while others form nonunions, and lead to development of novel therapeutics modulating immune cells, to increase fracture healing and prevent nonunions. Using novel femoral segmental and critical-size defect models in mice, we identified a systemic and significant increase in immature myeloid cell (IMC) infiltration during the initial phase of fracture healing until boney union is complete. Using gemcitabine to specifically ablate the IMC population, we confirmed delayed bone healing. Further, adoptive transfer of IMC increased bone growth in a nonunion model, signifying the role of this unique cell population in fracture healing. We also identified IMC post-fracture have the ability to increase endothelial cell migration, and tube formation, signaling the essential communication between the immune system and angiogenesis as a requirement for proper bone healing. Based on this data we propose that IMC may play a significant role in fracture healing and therapeutic targeting of IMC after fracture would minimize the chances of eventual nonunion pathology.

Acquired resistance to androgen receptor (AR)-targeted therapies compels the development of novel treatment strategies for castration-resistant prostate cancer (CRPC). Here, we report a profound effect of endostatin on prostate cancer cells by efficient intracellular trafficking, direct interaction with AR, reduction of nuclear AR level, and down-regulation of AR-target gene transcription. Structural modeling followed by functional analyses further revealed that phenylalanine-rich α1-helix in endostatin—which shares structural similarity with noncanonical nuclear receptor box in AR—antagonizes AR transcriptional activity by occupying the activation function (AF)-2 binding interface for coactivators and N-terminal AR AF-1. Together, our data suggest that endostatin can be recognized as an endogenous AR inhibitor that impairs receptor function through protein–protein interaction. These findings provide new insights into endostatin whose antitumor effect is not limited to inhibiting angiogenesis, but can be translated to suppressing AR-mediated disease progression in CRPC.

Anti-PD1 therapies show a remarkable improvement in response over many standard of care regimens, but there is a significant need to further increase patient responses. Recent clinical studies show improved response rate when combining anti-PD1 and anti-CTLA4 therapies. This provides “proof of concept” that the combination of two immunotherapies can enhance efficacy. The goal of our work was to evaluate the anti-tumor efficacy of B and T lymphocyte attenuator (BTLA) antagonism with anti-PD1 therapy in preclinical mouse tumor models. BTLA is an inhibitory co-receptor that modulates T cell function and is a marker of “exhausted” T cells. The inhibitory signal mediated by BTLA is initiated following engagement with herpesvirus entry mediator (HVEM), a ubiquitous receptor that is highly expressed on malignant cells. One of the challenges to evaluating BTLA in mouse models is the presence of strain-specific allelic polymorphisms. These sequence differences occur adjacent to the HVEM binding sites and can interfere with the ability of certain antibodies to recognize BTLA in different strains of mice or block the HVEM-BTLA signaling. C57BL/6-specific and strain agnostic BTLA binding antibodies are commercially available, but none antagonize HVEM signaling through BTLA in BALB/c mice. To more comprehensively evaluate the anti-BTLA/anti-PD1 combination regimen we generated a first-in-class BALB/c strain polymorphism specific BTLA antagonist monoclonal antibody (40E4) and identified clone PJ196 as a C57BL/6 strain polymorphism specific antagonist. We show that 40E4 and PJ196 not only block BTLA:HVEM interaction as measured by cell ELISA, but also reverse HVEM-mediated suppression in primary T cells from the appropriate mouse strain. An effector function reduced mouse IgG1 (D265A) chimeric form of 40E4 was generated for in vivo use. In combination with anti-PD1 (mDX400), 40E4 mIgG1 (D265A) shows improved tumor growth inhibition (TGI) over mDX400 alone in preclinical subcutaneous mouse models of breast cancer (EMT6) and colon cancer (CT26). To show that anti-BTLA enhancement of the anti-tumor response to mDX400 is not unique to BALB/c mice, we tested the combination of a C57BL/6 BTLA strain-polymorphism specific monoclonal antibody (PJ196) with mDX400. Using the subcutaneous MB49 model of bladder cancer, we show that treatment with PJ196 and mDX400 significantly improved TGI over mDX400 alone. This is the first report evaluating a BTLA antagonist monoclonal antibody in preclinical mouse tumor models and the first report to show BTLA antagonism enhances the effect of anti-PD1 therapy. Citation Format: Nathan R. Miselis, Douglas Linn, Clifford Restaino, Toya Baral, Jane Xia, Roanna Ueda, Anandi Sawant, Jeanne Baker, Gopalan Raghunathan, Xinzhong Wang1, Edward Bowman, Selvakumar Sukumar. Antagonism of the co-inhibitory receptor BTLA enhances efficacy of anti-PD-1 treatment in murine syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 577. doi:10.1158/1538-7445.AM2017-577

Blockade of the checkpoint inhibitor PD-1 has demonstrated remarkable success in the clinic for the treatment of a growing list of different cancers. However, several tumor types are resistant to anti-PD-1 monotherapy. This observation has spurred numerous combination studies to reveal what additional therapeutic interventions may complement anti-PD1 blockade. Recently it has been shown that immunogenic cell death (ICD), induced by radiation and/or chemotherapy, improves T cell responses against different tumor types. ICD is characterized by damage-associated molecular patterns (DAMPs) including surface expression of calreticulin, and release of ATP and HMGB1. Recognition of DAMPs triggers dendritic cell maturation and functions that are critical for tumor antigen-specific T cell activation. Thus therapies that evoke ICD may further augment anti-tumor immunity elicited by anti-PD-1. In mouse syngeneic tumor models, we observed combinatorial anti-tumor activity of anti-PD1 and the cyclin-dependent kinase inhibitor, dinaciclib. We hypothesized that dinaciclib potentiates the effects of anti-PD-1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib express the hallmarks of ICD including HMGB1 and ATP release and surface expression of calreticulin. Dinaciclib treatment also increases tumor cell phagocytosis and induces dendritic cell maturation. Furthermore, mice immunized with dinaciclib-treated tumor cells are resistant to subsequent tumor challenge. Tumors from mice receiving anti-PD1 and dinaciclib have increased T cell infiltration and dendritic cell activation, indicating the overall quality of the immune response generated may be improved with the combo. Taken together, these findings suggest a potential mechanism for the observed synergy between dinaciclib and anti-PD1. Dinaciclib induces immunogenic cell death, converting the tumor cell into an endogenous vaccine and thereby boosting the effects of anti-PD-1. Citation Format: Dewan Mohammed Sakib Hossain, Fernando Ugarte, Anandi Sawant, Mingmei Cai, Venkataraman Sriram, Elaine Pinheiro, Svetlana Sadekova, Alissa Chackerian. Dinaciclib induces immunogenic cell death and enhances anti-PD-1 mediated tumor suppression. [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 562.