IL1R2 Blockade Alleviates Immunosuppression and Potentiates Anti-PD-1 Efficacy in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. IL1 receptor type 2 (IL1R2) promotes breast tumor-initiating cell (BTIC) self-renewal and tumor growth in TNBC, indicating that targeting it could improve patient treatment. In this study, we observed that IL1R2 blockade strongly attenuated macrophage recruitment and the polarization of tumor-associated macrophages (TAM) to inhibit BTIC self-renewal and CD8+ T-cell exhaustion, which resulted in reduced tumor burden and prolonged survival in TNBC mouse models. IL1R2 activation by TAM-derived IL1ß increased PD-L1 expression by interacting with the transcription factor Yin Yang 1 (YY1) and inducing YY1 ubiquitination and proteasomal degradation in both TAMs and TNBC cells. Loss of YY1 alleviated the transcriptional repression of c-Fos, which is a transcriptional activator of PDL-1. Combined treatment with an IL1R2-neutralizing antibodies and anti-PD-1 led to enhanced antitumor efficacy and reduced TAMs, BTICs, and exhausted CD8+ T cells. These results suggest that IL1R2 blockade might be a strategy to potentiate immune checkpoint blockade efficacy in TNBC to improve patient outcomes. Significance: IL1R2 in both macrophages and breast cancer cells orchestrates an immunosuppressive tumor microenvironment by upregulating PD-L1 expression and can be targeted to enhance the efficacy of anti-PD-1 in triple-negative breast cancer.

Breast Tumor Cells Highly Resistant to Drugs Are Controlled Only by the Immune Response Induced in an Immunocompetent Mouse Model.

BACKGROUND: The tumor cells responsible for metastasis are highly resistant to chemotherapy and have characteristics of stem cells, with a high capacity for self-regeneration and the use of detoxifying mechanisms that participate in drug resistance. In vivo models of highly resistant cells allow us to evaluate the real impact of the immune response in the control of cancer. MATERIALS AND METHODS: A tumor population derived from the 4T1 breast cancer cell line that was stable in vitro and highly aggressive in vivo was obtained, characterized, and determined to exhibit cancer stem cell (CSC) phenotypes (CD44+, CD24+, ALDH+, Oct4+, Nanog+, Sox2+, and high self-renewal capacity). Orthotopic transplantation of these cells allowed us to evaluate their in vivo susceptibility to chemo and immune responses induced after vaccination. RESULTS: The immune response induced after vaccination with tumor cells treated with doxorubicin decreased the formation of tumors and macrometastasis in this model, which allowed us to confirm the immune response relevance in the control of highly chemotherapy-resistant ALDH+ CSCs in an aggressive tumor model in immunocompetent animals. CONCLUSIONS: The antitumor immune response was the main element capable of controlling tumor progression as well as metastasis in a highly chemotherapy-resistant aggressive breast cancer model.

Anti-CD123 chimeric antigen receptor T-cells (CART): an evolving treatment strategy for hematological malignancies, and a potential ace-in-the-hole against antigen-negative relapse.

Chimeric antigen receptor-modified T-cells (CART) are a potent and targeted immunotherapy which have induced remissions in some patients with chemotherapy refractory or relapsed (RR) hematologic malignancies. Hundreds of patients have now been treated worldwide with anti-CD19 CART cells, with complete response rates of up to 90%. CART therapy has a unique toxicity profile, and unfortunately not all responses are durable. Treatment failure occurs via two main routes - by loss of the CART cell population, or relapse with antigen loss. Emerging data indicate that targeting an alternative antigen instead of, or as well as CD19, could improve CART cell efficacy and reduce antigen-negative relapse. Other strategies include the addition of other immune-based therapies. This review explores the rationale, pre-clinical data and currently investigative strategies underway for CART therapy targeting the myeloid and lymphoid stem/progenitor antigen CD123.

Breast cancer stem-like cells can promote metastasis by activating platelets and down-regulating antitumor activity of natural killer cells.

OBJECTIVE: To investigate whether cancer stem cells (CSCs) more efficiently activating platelets and evading immune surveillance than non-CSCs thus promoting metastasis. METHODS: We enriched and identified sphere-forming cells (SFCs) and coincubated washed platelets with several platelet activators including collagen, 4T1 and SFCs. Platelet-coating tumor cells, platelet activation and TGF-ß1 release were analyzed. Then natural kell cells (NK) were incubated with supernatants of different activated platelet samples what we called sample release (SR). The degranulation assay and NKG2D expression on NK cells were conducted by flow cytometry. Finally tissue factor (TF) expression of SFCs or 4T1 were evaluated by western blot. RESULTS: Breast cancer cell line 4T1 could form spheres in serum-free medium at low adherence. Sphere-forming cells expressed high levels of the CD24-/lowCD44 + stem cell phenotype. Both sphere-forming cells or 4T1 were coated with abundant platelets while sphere-forming cells induced significantly higher expression of platelet activating receptor CD62p than 4T1 did (P < 0.01). And sphere-forming cells induced platelets to produce more TGF-ß1 than 4T1 did (P < 0.01). Furthermore, sample releases induced by sphere-forming cells caused more vigorous inhibition of NK cells antitumor reactivity (P < 0.05) and reduced NKG2D expression (P < 0.01). The final results showed that sphere-forming cells expressed higher levels of TF than 4T1 (P < 0.05). CONCLUSION: Our findings indicate that CSCs could efficiently activate platelets, induce platelets to secrete more TGF-ß1, decrease NKG2D expression and inhibit antitumor activity of NK cell, compared with 4T1. And higher levels of TF expression of CSCs may account for this correlation of CSCs and platelets.

Amarogentin regulates self renewal pathways to restrict liver carcinogenesis in experimental mouse model.

Amarogentin, a secoiridoid glycoside isolated from medicinal plant Swertia chirata, was found to restrict CCl4 /N-nitrosodiethyl amine (NDEA) induced mouse liver carcinogenesis by modulating G1/S cell cycle check point and inducing apoptosis. To understand its therapeutic efficacy on stem cell self renewal pathways, prevalence of CD44 positive cancer stem cell (CSC) population, expressions (mRNA/protein) of some key regulatory genes of self renewal Wnt and Hedgehog pathways along with expressions of E-cadherin and EGFR were analyzed during the liver carcinogenesis and in liver cancer cell line HepG2. It was observed that amarogentin could significantly reduce CD44 positive CSCs in both pre and post initiation stages of carcinogenesis than carcinogen control mice. In Wnt pathway, amarogentin could inhibit expressions of ß-catenin, phospho ß-catenin (Y-654) and activate expressions of antagonists sFRP1/2 and APC in the liver lesions. In Hedgehog pathway, decreased expressions of Gli1, sonic hedgehog ligand, and SMO along with up-regulation of PTCH1 were seen in the liver lesions due to amarogentin treatment. Moreover, amarogentin could up-regulate E-cadherin expression and down-regulate expression of EGFR in the liver lesions. Similarly, amarogentin could inhibit HepG2 cell growth along with expression and prevalence of CD44 positive CSCs. Similar to in vivo analysis, amarogentin could modulate the expressions of the key regulatory genes of the Wnt and hedgehog pathways and EGFR in HepG2 cells. Thus, our data suggests that the restriction of liver carcinogenesis by amarogentin might be due to reduction of CD44 positive CSCs and modulation of the self renewal pathways. © 2015 Wiley Periodicals, Inc.

Immunotargeting of Antigen xCT Attenuates Stem-like Cell Behavior and Metastatic Progression in Breast Cancer.

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2(+) breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc(-), as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy.

Enhancement of cancer stem cell susceptibility to conventional treatments through complementary yoga therapy: possible cellular and molecular mechanisms.

Cancer stem cells (CSCs) are stem-like tumor populations that are reported to contribute towards tumor growth, maintenance and recurrence after therapy. Hypoxia increases CSC fraction and promotes acquisition of a stem-cell-like state. Cancer stem cells are critically dependant on the hypoxia-inducible factor-1 (HIF-1) for survival, self-renewal, tumor growth and maintenance of their undifferentiated phenotype. Recent researches show that stage of differentiation of the tumor cells is predictive of their susceptibility to natural killer cell (NK) cell mediated cytotoxicity and cancer stem cells are significant targets of NK cell cytotoxicity. Studies also show that reversion of tumor cells to a less-differentiated phenotype can be achieved by blocking NFκB. Yoga therapy (yogic lifestyle modifications encompassing physical postures, breathing practices, relaxation techniques and meditations) is known to modulate neural, endocrine and immune functions at the cellular level through influencing cell cycle control, aging, oxidative stress, apoptosis and several pathways of stress signaling molecules. Yoga therapy has also been shown to enhance natural killer cell activity and modulate stress and DNA damage in breast cancer patients receiving radiotherapy. Recent study found that brief daily yogic meditation may reverse the pattern of increased NFκB-related transcription of pro-inflammatory cytokines in leukocytes. Thus, yoga therapy has the potential to reduce cancer stem cell survival, self -renewal and tumor growth by modifying the tumor micro-environment through various mechanisms such as; 1) reducing HIF-1 activity by enhanced oxygenation, 2) promoting NK cell activity directly (or indirectly through down regulating NFκB expression), thereby enhancing NK cell mediated CSC lysis, and 3) by minimizing the aberrant expressions or activities of various hormones, cytokines, chemokines and tumor signaling pathways. Yoga therapy may have a synergistic effect with conventional modalities of treatment in preventing cancer progression and recurrences.

Design of clinical trials for therapeutic cancer vaccines development.

Advances in molecular and cellular biology as well as biotechnology led to definition of a group of drugs referred to as medicinal products of advanced technologies. It includes gene therapy products, somatic cell therapeutics and tissue engineering. Therapeutic cancer vaccines including whole cell tumor cells vaccines or gene modified whole cells belong to somatic therapeutics and/or gene therapy products category. The drug development is a multistep complex process. It comprises of two phases: preclinical and clinical. Guidelines on preclinical testing of cell based immunotherapy medicinal products have been defined by regulatory agencies and are available. However, clinical testing of therapeutic cancer vaccines is still under debate. It presents a serious problem since recently clinical efficacy of the number of cancer vaccines has been demonstrated that focused a lot of public attention. In general clinical testing in the current form is very expensive, time consuming and poorly designed what may lead to overlooking of products clinically beneficial for patients. Accordingly regulatory authorities and researches including Cancer Vaccine Clinical Trial Working Group proposed three regulatory solutions to facilitate clinical development of cancer vaccines: cost-recovery program, conditional marketing authorization, and a new development paradigm. Paradigm includes a model in which cancer vaccines are investigated in two types of clinical trials: proof-of-principle and efficacy. The proof-of-principle trial objectives are: safety; dose selection and schedule of vaccination; and demonstration of proof-of-principle. Efficacy trials are randomized clinical trials with objectives of demonstrating clinical benefit either directly or through a surrogate. The clinical end points are still under debate.

Cutaneous T cell lymphoma: translating immunobiology into therapeutic opportunities.

Cutaneous T cell lymphoma (CTCL) has always served as a proving ground where conceptual advances in immunology can be tested and the results translated into clinical practice. From the earliest studies that used sheep red blood cells to identify the malignant cell as a T lymphocyte to molecular demonstration of the clonalilty of the disease, basic science techniques have provided sign posts that allow us to understand the clinical features seen in the patients. We continue to apply this paradigm to develop new insights into the role of the immune system in CTCL with the goal of using this knowledge to enhance the therapeutic options available to the patient. This article will review the studies that have led to our current understanding of the immunobiology of CTCL and the new therapeutic approaches that are being tested in this disease.

Isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma.

AIM: To explore the method of isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma cell line PANC-1. METHODS: The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 (1:1 volume) (DMEM-F12) supplemented with 20% fetal bovine serum (FBS). Subpopulation cells with properties of tumor stem cells were isolated from pancreatic adenocarcinoma cell line PANC-1 according to the cell surface markers CD44 and CD24 by flow cytometry. The proliferative capability of these cells in vitro were estimated by 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) method. And the tumor growth of different subpopulation cells which were injected into the hypodermisof right and left armpit of nude mice was studied, and expression of CD44 and CD24 of the CD44(+)CD24(+) cell-formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex (ABC) immunohistochemical staining. RESULTS: The 5.1%-17.5% of sorted PANC-1 cells expressed the cell surface marker CD44, 57.8% -70.1% expressed CD24, only 2.1%-3.5% of cells were CD44(+) CD24(+). Compared with CD44(-)CD24(-) cells, CD44(+)CD24(+) cells had a lower growth rate in vitro. Implantation of 10(4) CD44(-)CD24(-) cells in nude mice showed no evident tumor growth at wk 12. In contrast, large tumors were found in nude mice implanted with 10(3) CD44(+)CD24(+) cells at wk 4 (2/8), a 20-fold increase in tumorigenic potential (P < 0.05 or P < 0.01). There was no obvious histological difference between the cells of the CD44(+)CD24(+) cell-formed nodules and PANC-1 cells. CONCLUSION: CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44(+)CD24(+) have properties of tumor stem cells. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, it may be a very promising target for new drug development.

The effect of tetraethylenepentamine, a synthetic copper chelating polyamine, on expression of CD34 and CD38 antigens on normal and leukemic hematopoietic cells.

We have previously found that the synthetic polyamine tetraethylenepentamine (TEPA) significantly delayed differentiation and prolonged expansion of cord-blood derived HPC in cytokine-supplemented cultures. Most HPC have the CD34+CD38+ phenotype, but the minority CD34+38- cells are primitive subset of HPC that have the potential for long-term repopulation in vivo. We investigated the effect of TEPA on the CD34/CD38 surface antigen expression of human myeloid leukemia cell lines as well as normal cord blood derived hematopoietic cells. Confirming previous results, our data showed that both the leukemic and normal cells increased their CD38 expression when grown in serum-containing medium or when treated with retinoic acid. In the present study, we found that TEPA inhibited CD38 under these conditions in both normal and leukemic cells. As for CD34, TEPA increased the proportion of CD34 cells in short- and long-term normal cultures but not in the leukemic cell lines. These results suggest that ex vivo expansion of HPC depends on the presence of CD34+CD38- cells and that TEPA prolongs HPC expansion by inhibiting the CD38- to CD38+ transition.

Preclinical evaluation of immunoconjugates consisting of doxorubicin linked to complement-fixing monoclonal antibody DLC-48 for bone marrow purging of B-cell lymphomas.

Complement-fixing monoclonal antibody DLC-48 was linked covalently to doxorubicin using two methodologies, and the resulting conjugates were evaluated for their usefulness in purging B-cell lymphomas from human bone marrow autografts using a clonogenic assay for the large cell lymphoma cell line SU-DHL-4. Conjugation did not impair the reactivity of the antibodies. The resultant molar ratios provided for maximal immunoreactivity at doxorubicin concentrations that would permit the growth of CFU-GM. Conjugate DLC-48D-G retained its complement-fixing capacity, and was more cytotoxic to SU-DHL-4 cells, even in the absence of complement, than equivalent doses of doxorubicin. This effect appeared to be non-specific, in that CFU-GM were eliminated, even at low concentrations. DLC-48D-G was no more effective than equivalent doses of doxorubicin, and equally cytotoxic to CFU-GM. Mixtures of unconjugated DLC-48 and doxorubicin, however, were more effective than doxorubicin alone. The mechanism of action of these conjugates is under study. Alternative methods of conjugation will be investigated.