Bortezomib Augments Natural Killer Cell Targeting of Stem-Like Tumor Cells.

Tumor cells harboring stem-like/cancer stem cell (CSC) properties have been identified and isolated from numerous hematological and solid malignancies. These stem-like tumor cells can persist following conventional cytoreductive therapies, such as chemotherapy and radiotherapy, thereby repopulating the tumor and seeding relapse and/or metastasis. We have previously shown that natural killer (NK) cells preferentially target stem-like tumor cells via non- major histocompatibility complex (MHC) restricted mechanisms. Here, we demonstrated that the proteasome inhibitor, bortezomib, augments NK cell targeting of stem cell-like tumor cells against multiple solid human tumor-derived cancer lines and primary tissue samples. Mechanistically, this was mediated by the upregulation of cell surface NK ligands MHC class I chain-related protein A and B (MICA and MICB) on aldehyde dehydrogenases (ALDH)-positive CSCs. The increased expression of MICA and MICB on CSC targets thereby enhanced NK cell mediated killing in vitro and ex vivo from both human primary tumor and patient-derived xenograft samples. In vivo, the combination of bortezomib and allogeneic NK cell adoptive transfer in immunodeficient mice led to increased elimination of CSCs as well as tumor growth delay of orthotopic glioblastoma tumors. Taken together, our data support the combination bortezomib and NK transfer as a strategy for both CSC targeting and potentially improved outcomes in clinical cancer patients

Anti-proliferative but not anti-angiogenic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue sarcoma.

BackgroundIncreasing studies implicate cancer stem cells (CSCs) as the source of resistance and relapse following conventional cytotoxic therapies. Few studies have examined the response of CSCs to targeted therapies, such as tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have differential effects on CSC populations depending on their mechanism of action (anti-proliferative vs. anti-angiogenic).MethodsWe exposed human sarcoma cell lines to sorafenib, regorafenib, and pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray (TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and non-parametric statistical analyses were performed as appropriate.ResultsAfter functionally validating the CSC phenotype of ALDHbright sarcoma cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell lines (P < 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright cells from baseline (P < 0.05). In contrast, we observed negligible effects on viability and CSC sub-populations with pazopanib. At low doses, there was progressive CSC enrichment in vitro after longer term exposure to sorafenib although the anti-proliferative effects were attenuated. In vivo, sorafenib improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells 2.5 - 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib and regorafenib with a corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects from pazopanib were observed. TMA analysis of archived specimens from sarcoma patients treated with sorafenib demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas clinical specimens obtained longitudinally from a patient treated with pazopanib showed no enrichment for ALDHbright cells (P > 0.05).ConclusionsAnti-proliferative TKIs appear to enrich for sarcoma CSCs while anti-angiogenic TKIs do not. The rational selection of targeted therapies for sarcoma patients may benefit from an awareness of the differential impact of TKIs on CSC populations

Anti-proliferative but not anti-angiogenic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue sarcoma

The templated zeolite-analogue GaPO-34 (CHA structure type) crystallises from a gel precursor Ga2O3:2H3PO4:1HF:1.7SDA:70H2O (where SDA = structure directing agent), treated hydrothermally for 24 hours at 170 °C using either pyridine or 1-methylimizadole as SDA and one of either poorly crystalline ε-Ga2O3 or γ-Ga2O3 as gallium precursor. If the same gels are stirred for periods shorter than 2 hours but treated under identical hydrothermal conditions, then a second phase crystallises, free of GaPO-34. If β-Ga2O3 is used as a reagent only the second phase is found to crystallise, irrespective of gel aging time. The competing phase, which we denote GaPO-34A, has been structurally characterised using synchrotron powder X-ray diffraction for the pyridine material, GaPO-34A(pyr), and using single-crystal X-ray diffraction for the 1-methylimiazole material, GaPO-34A(mim). The structure of GaPO-34A(pyr), P1 @#x0305;, a = 10.22682(6) Å, b = 12.09585(7) Å, c = 13.86713(8) Å, α = 104.6531(4) °, β = 100.8111(6) °, γ = 102.5228(6) °, contains 7 unique gallium sites and 6 phosphorus sites, with empirical formula [Ga7P6O24(OH)2F3(H2O)2].2(C5NH6). GaPO-34A(mim) is isostructural but is modelled as a half volume unit cell, P1 @#x0305;, a = 5.0991(2) Å, b = 12.0631(6) Å, c = 13.8405(9) Å, α = 104.626(5) °, β = 100.346(5) °, γ = 101.936(4) °, with a gallium and a bridging fluoride partially occupied and two partially occupied SDA sites. Solid-state 31P and 71Ga NMR spectroscopy confirms the structural complexity of GaPO-34A with signals resulting from overlapping lineshapes from multiple Ga and P sites, while 1H and 13C solid-state NMR spectra confirm the presence of the protonated SDA and provide evidence for disorder in the SDA. The protonated SDA is located in 14-ring one-dimensional channels with hydrogen bonding deduced from the SDA nitrogens to framework oxygen distances. Upon thermal treatment to investigate SDA removal, structure collapse occurs, which may be due the large number of bridging hydroxides and fluorides in the as-made material, and the unequal amounts of gallium and phosphorus present

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Natural killer cell immunotherapy to target stem-like tumor cells.

Advances in cancer immunotherapy are leading to its increasing and successful application for the treatment of solid-tissue cancers. Despite the recent advances there are still significant barriers, in particular, evidence for significant tumor heterogeneity, both genetic and epigenetic that limit long-term efficacy. Subpopulations of "stem-like" tumor cells have been identified in nearly all human malignancies based on both morphologic and functional criteria. Also called cancer stem cells or CSCs, these quiescent cells display enhanced tumorigenic potential and are capable of repopulating tumors in the wake of traditional cytoreductive therapies. These CSCs may be best targeted via immunotherapy. Our lab has identified activated natural killer (NK) cell-based therapy as an effective method to target CSCs particularly after radiation therapy for solid tumors. Using a variety of in vitro and in vivo methods, including the utilization of primary tumor tissue and patient-derived xenografts, we observed that autologous and allogeneic NK cells possess the ability to&nbsp;preferentially kill stem-like cells or CSCs from freshly isolated patient samples representing a broad spectrum of tumor types, including pancreatic cancers, breast cancers, and sarcomas. The results indicated that CSCs express stress ligand molecules capable of being targeted by NKG2D on NK cells and that prior radiation therapy can both deplete the cycling non-CSCs bulk tumor population and upregulate these stress ligands on the CSC making this an effective combination approach

Leveraging natural killer cells for cancer immunotherapy

Natural killer (NK) cells are potent antitumor effector cells of the innate immune system. Based on their ability to eradicate tumors in vitro and in animal models, significant enthusiasm surrounds the prospect of leveraging human NK cells as vehicles for cancer immunotherapy. While interest in manipulating the effector functions of NK cells has existed for over 30 years, there is renewed optimism for this approach today. Although T cells receive much of the clinical and preclinical attention when it comes to cancer immunotherapy, new strategies are utilizing adoptive NK-cell immunotherapy and monoclonal antibodies and engineered molecules which have been developed to specifically activate NK cells against tumors. Despite the numerous challenges associated with the preclinical and clinical development of NK cell-based therapies for cancer, NK cells possess many unique immunological properties and hold the potential to provide an effective means for cancer immunotherapy

Natural killer cell immunotherapy to target stem-like tumor cells.

Advances in cancer immunotherapy are leading to its increasing and successful application for the treatment of solid-tissue cancers. Despite the recent advances there are still significant barriers, in particular, evidence for significant tumor heterogeneity, both genetic and epigenetic that limit long-term efficacy. Subpopulations of "stem-like" tumor cells have been identified in nearly all human malignancies based on both morphologic and functional criteria. Also called cancer stem cells or CSCs, these quiescent cells display enhanced tumorigenic potential and are capable of repopulating tumors in the wake of traditional cytoreductive therapies. These CSCs may be best targeted via immunotherapy. Our lab has identified activated natural killer (NK) cell-based therapy as an effective method to target CSCs particularly after radiation therapy for solid tumors. Using a variety of in vitro and in vivo methods, including the utilization of primary tumor tissue and patient-derived xenografts, we observed that autologous and allogeneic NK cells possess the ability to preferentially kill stem-like cells or CSCs from freshly isolated patient samples representing a broad spectrum of tumor types, including pancreatic cancers, breast cancers, and sarcomas. The results indicated that CSCs express stress ligand molecules capable of being targeted by NKG2D on NK cells and that prior radiation therapy can both deplete the cycling non-CSCs bulk tumor population and upregulate these stress ligands on the CSC making this an effective combination approach

Immune targeting of cancer stem cells in gastrointestinal oncology.

The cancer stem cell (CSC) hypothesis postulates that a sub-population of quiescent cells exist within tumors which are resistant to conventional cytotoxic/anti-proliferative therapies. It is these CSCs which then seed tumor relapse, even in cases of apparent complete response to systemic therapy. Therefore, therapies, such as immunotherapy, which add a specific anti-CSC strategy to standard cytoreductive treatments may provide a promising new direction for future cancer therapies. CSCs are an attractive target for immune therapies since, unlike chemotherapy or radiotherapy, immune effector cells do not specifically require target cells to be proliferating in order to effectively kill them. Although recent advances have been made in the development of novel systemic and targeted therapies for advanced gastro-intestinal (GI) malignancies, there remains an unmet need for durable new therapies for these refractory malignancies. Novel immunotherapeutic strategies targeting CSCs are in pre-clinical and clinical development across the spectrum of the immune system, including strategies utilizing adaptive immune cell-based effectors, innate immune effectors, as well as vaccine approaches. Lastly, since important CSC functions are affected by the tumor microenvironment, targeting of both cellular (myeloid derived suppressor cells and tumor-associated macrophages) and sub-cellular (cytokines, chemokines, and PD1/PDL1) components of the tumor microenvironment is under investigation in the immune targeting of CSCs. These efforts are adding to the significant optimism about the potential utility of immunotherapy to overcome cancer resistance mechanisms and cure greater numbers of patients with advanced malignancy

Models to Study NK Cell Biology and Possible Clinical Application

Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. NK cells rapidly recognize and respond to abnormal cells in the absence of prior sensitization due to their wide array of germline-encoded inhibitory and activating receptors, which differs from the receptor diversity found in B and T lymphocytes resulting from the use of recombination-activation gene (RAG) enzymes. Although NK cells have traditionally been described as natural killers that provide a first line of defense prior to the induction of adaptive immunity, a more complex view of NK cells is beginning to emerge indicating they may also function in various immunoregulatory roles and have the capacity to shape adaptive immune responses. With the growing appreciation for the diverse functions of NK cells and recent technological advancements that allow for a more in-depth understanding of NK cell biology, we can now begin to explore new ways to manipulate NK cells to increase their clinical utility. In this overview unit, we introduce the reader to various aspects of NK cell biology by reviewing topics ranging from NK cell diversity and function, mouse models and the roles of NK cells in health and disease, to potential clinical applications

Targeting Cancer Stem Cells with Natural Killer Cell Immunotherapy.

IntroductionStandard cytoreductive cancer therapy, such as chemotherapy and radiotherapy, are frequently resisted by a small portion of cancer cells with 'stem-cell' like properties including quiescence and repopulation. Immunotherapy represents a breakthrough modality for improving oncologic outcomes in cancer patients. Since the success of immunotherapy is not contingent on target cell proliferation, it may also be uniquely suited to address the problem of resistance and repopulation exerted by cancer stem cells (CSCs). Areas covered: Natural killer (NK) cells have long been known for their ability to reject allogeneic hematopoietic stem cells, and there are increasing data demonstrating that NK cells can selectively identify and lyse CSCs. The authors review the current knowledge of CSCs and NK cells and highlight recent studies that support the concept that NK cells are capable of targeting CSC in solid tumors, especially in the context of combination therapy simultaneously targeting non-CSCs and CSCs. Expert opinion: Unlike cytotoxic cancer treatments, NK cells can target and eliminate quiescent/non-proliferating cells such as CSCs, and these enigmatic cells are an important source of relapse and metastasis. NK targeting of CSCs represents a novel and potentially high impact method to capitalize on the intrinsic therapeutic potential of NK cells