The CRISPR/Cas System in Human Cancer.

The use of CRISPR as a genetic editing tool modified the oncology field from its basic to applied research for opening a simple, fast, and cheaper way to manipulate the genome. This chapter reviews some of the major uses of this technique for in vitro- and in vivo-based biological screenings, for cellular and animal model generation, and new derivative tools applied to cancer research. CRISPR has opened new frontiers increasing the knowledge about cancer, pointing to new solutions to overcome several challenges to better understand the disease and design better treatments.

The ambiguous role of obesity in oncology by promoting cancer but boosting antitumor immunotherapy.

Obesity is nowadays considered a pandemic which prevalence's has been steadily increasingly in western countries. It is a dynamic, complex, and multifactorial disease which propitiates the development of several metabolic and cardiovascular diseases, as well as cancer. Excessive adipose tissue has been causally related to cancer progression and is a preventable risk factor for overall and cancer-specific survival, associated with poor prognosis in cancer patients. The onset of obesity features a state of chronic low-grade inflammation and secretion of a diversity of adipocyte-derived molecules (adipokines, cytokines, hormones), responsible for altering the metabolic, inflammatory, and immune landscape. The crosstalk between adipocytes and tumor cells fuels the tumor microenvironment with pro-inflammatory factors, promoting tissue injury, mutagenesis, invasion, and metastasis. Although classically established as a risk factor for cancer and treatment toxicity, recent evidence suggests mild obesity is related to better outcomes, with obese cancer patients showing better responses to treatment when compared to lean cancer patients. This phenomenon is termed obesity paradox and has been reported in different types and stages of cancer. The mechanisms underlying this paradoxical relationship between obesity and cancer are still not fully described but point to systemic alterations in metabolic fitness and modulation of the tumor microenvironment by obesity-associated molecules. Obesity impacts the response to cancer treatments, such as chemotherapy and immunotherapy, and has been reported as having a positive association with immune checkpoint therapy. In this review, we discuss obesity's association to inflammation and cancer, also highlighting potential physiological and biological mechanisms underlying this association, hoping to clarify the existence and impact of obesity paradox in cancer development and treatment.

The prevalence and prognostic impact of tumor-infiltrating lymphocytes in uterine carcinosarcoma.

OBJECTIVE: To examine the prevalence and prognostic role of tumor microenvironment (TME) markers in uterine carcinosarcoma (UCS) through immunohistochemical characterization. METHODS: The internal database of our institution was queried out for women with UCS who underwent surgery and thereafter postoperative chemotherapy with carboplatin and paclitaxel between January 2012 and December 2017. Tissue microarrays containing surgical samples of UCS from 57 women were assessed by immunohistochemistry for CD3, CD4, CD8, FOXP3, PD-1, PD-L1, and PD-L2. RESULTS: The mean age was 65.3 years (range, 49 to 79 years). For the epithelial component (E), CD3_E and CD4_E were highly expressed in 38 (66.7%) and in 40 (70.1%) patients, respectively, and were significantly associated with more advanced stages (p = 0.038 and p = 0.025, respectively). CD8_E was highly expressed in 42 (73.7%) patients, FOXP3_E 16 (28.1%), PD-1_E 35 (61.4%), PD-L1_E 27 (47.4%) and PD-L2_E 39 (68.4%). For the sarcomatous component (S), the prevalence of high expression was: CD3_S 6 (10.5%), CD4_S 20 (35.1%), CD8_S 44 (77.2%), FOXP3_S 8 (14%), PD-1_S 14 (24.6%), PD-L1_S 14 (24.6%) and PD-L2_S 8 (14%). By multivariate analysis, the CD8/FOXP3_S ratio (p = 0.026), CD4_E (p = 0.010), PD-L1_E (p = 0.013) and PD-L1_S (p = 0.008) markers significantly influenced progression-free survival. CD4/FOXP3_S ratio (p = 0.043), PD-1_E (p = 0.011), PD-L1_E (p = 0.036) and PD-L1_S (p = 0.028) had a significant association with overall survival. CONCLUSION: Some differences in UCS clinical outcomes may be due to the subtype of TILs and PD-1/PD-L1 axis immune checkpoint signaling.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VII. Present and future of technologies for production of CAR cell therapies.

Chimeric Antigen Receptor T (CAR-T) cells are certainly an important therapy for patients with relapsed and/or refractory hematologic malignancies. Currently, there are five CAR-T cell products approved by the FDA but several research groups and/or biopharmaceutical companies are encouraged to develop new products based on CAR cells using T or other cell types. Production of CAR cells requires intensive work from the basic, pre-clinical to translational levels, aiming to overcome technical difficulties and failure in the production. At least five key common steps are needed for the manipulation of T-lymphocytes (or other cells), such as: cell type selection, activation, gene delivery, cell expansion and final product formulation. However, reproducible manufacturing of high-quality clinical-grade CAR cell products is still required to apply this technology to a greater number of patients. This chapter will discuss the present and future development of new CAR designs that are safer and more effective to improve this therapy, achieving more selective killing of malignant cells and less toxicity to be applied in the clinical setting.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VIII: CAR-T cells: preclinical development - Safety and efficacy evaluation.

Currently, there are four CAR-T products commercially available on the market. CAR-T cells have shown high remission rates and they represent an effective treatment option for patients with resistant or refractory B cell malignancies. Approval of these cell therapy products came after an extended period of preclinical evaluation that demonstrated unprecedented efficacy in this difficult-to-treat patient population. This review article outlines the main preclinical evaluations needed for CAR T cell product development.

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms.

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit-megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

CAR-T cells leave the comfort zone: current and future applications beyond cancer.

Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in the immunotherapy field and has achieved great success following its approval in 2017 for the treatment of B cell malignancies. While CAR-T cells are mostly applied as anti-tumor therapy in the present, their initial concept was aimed at a more general purpose of targeting membrane antigens, thus translating in many potential applications. Since then, several studies have assessed the use of CAR-T cells toward non-malignant pathologies such as autoimmune diseases, infectious diseases and, more recently, cardiac fibrosis, and cellular senescence. In this review, we present the main findings and implications of CAR-based therapies for non-malignant conditions.

CRISPR / Cas-9 Vector System: Targets UL-39 and Inhibits HSV-1 Replication in Vitro

HSV-1 affects approximately 67% of the world population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyzes revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.

High infiltration of B cells in tertiary lymphoid structures, TCR oligoclonality, and neoantigens are part of esophageal squamous cell carcinoma microenvironment.

Esophageal squamous cell carcinoma (ESCA) exhibits high intratumoral molecular heterogeneity posing a challenge to cancer therapy. Immune checkpoint blockade therapy has been approved for this disease, but with modest results. RNA-Seq data from paired tumor and surrounding nonmalignant tissue from 14 patients diagnosed with ESCA without previous treatment and from The Cancer Genome Atlas-ESCA cohort were analyzed. Herein, we investigated ESCA immune landscape including mutation-derived neoantigens and immune cell subpopulations. Tumor-associated antigen expression was determined by in silico analyses and confirmed by immunohistochemistry showing that PRAME, CEACAM4, and MAGEA11 proteins are expressed on tumors. Immune checkpoint molecules gene expression was higher in the tumor compared with surrounding nonmalignant tissue, but its expression varies greatly among patients. TCR repertoire and BCR transcripts analysis evidenced low clonal diversity with one TCR clone predicted to be specific for a MAGEA11-derived peptide. A high number of B-cell clones infiltrating the tumors and the abundance of these cells in tertiary lymphoid structures observed in ESCA tumors support B cells as a potential immune modulator in this tumor.

Development of CAR-T cell therapy for B-ALL using a point-of-care approach.

Recently approved by the FDA and European Medicines Agency, CAR-T cell therapy is a new treatment option for B-cell malignancies. Currently, CAR-T cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs, and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CAR-T cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CAR-T cells can be generated and used without the need for ex vivo activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CAR-T cells are effective in vivo against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 h of gene transfer. In an effort to better characterize the infused CAR-T cells, we show that 19BBz T lymphocytes infused after 24 h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity in vivo. Our data suggest that POC approach is a viable alternative for the generation and use of CAR-T cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries.

TET2 haploinsufficiency alters reprogramming into induced pluripotent stem cells.

The discovery of the Ten-Eleven Translocation (TET) protein family was initiated by the identification of the MLL partner TET1, and of mutations in the TET2 gene in hematological malignancies including myeloproliferative neoplasms (MPN). TET1, 2 and 3 proteins hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Previous studies highlight the involvement of TET proteins in somatic cells reprogramming into induced pluripotent stem cells (iPSC), particularly Tet1 and 2 in mouse and TET1 in human. Here, we asked whether endogenous TET2 knockdown also displays this function. Using different shRNA against TET2, we provide evidence that TET2 strongly decreases the reprogramming of human hematopoietic progenitor cells into iPSC. Importantly, using 2 MPN patients, we observed that TET2 mutations affecting catalytic domain allowed iPSC generation. Instead, using another TET2 and TET3-mutated patient, we could only reprogram IPSC with TET3 mutation alone, suggesting that the type of TET2 mutation and/or the cooperation with TET3 mutations may alter the reprogramming activity. Altogether, this work highlights the importance of endogenous TET in the reprogramming process of human hematopoietic progenitors.

Transposon-mediated generation of CAR-T cells shows efficient anti B-cell leukemia response after ex vivo expansion.

CAR-T-cell therapy has shown considerable advance in recent years, being approved by regulatory agencies in US, Europe, and Japan for the treatment of refractory patients with CD19+ B-cell leukemia or diffuse large B-cell lymphoma. Current methods for CAR-T-cell production use viral vectors for T-cell genetic modification and can take up to 15 days to generate the infusion product. The development of simple and less costly manufacturing protocols is needed in order to meet the increasing demand for this therapy. In this present work, we generated 19BBz CAR-T cells in 8 days using a protocol based on the non-viral transposon-based vector Sleeping Beauty. The expanded cells display mostly a central memory phenotype, expressing higher levels of inhibitory receptors when compared with mock cells. In addition, CAR-T cells were cytotoxic against CD19+ leukemia cells in vitro and improved overall survival rates of mice xenografted with human RS4;11 or Nalm-6 B-cell leukemias. Infused CAR-T cells persisted for up to 28 days, showing that they are capable of long-term persistence and antitumor response. Altogether, these results demonstrate the effectiveness of our protocol and pave the way for a broader application of CAR-T-cell therapy.

CRISPR/Cas 9 system for the treatment of dilated cardiomyopathy: A hypothesis related to function of a MAP kinase.

Dilated cardiomyopathy (DCM) is a disease with high incidence and mortality rates. Its therapies have one primary goal, which is to minimize symptoms and it has only one effective approach to healing, the heart transplantation. As it is widely associated with genetic causes, the use of gene therapies, such as the CRISPR/Cas9 system, is a promising alternative to treat DCM. For this purpose, it is necessary to analyze possible target genes for this approach and what would be the implications of their use. Here, we hypothesized that cardiac troponin I type 3 interacting kinase (TNI3K), involved with superoxide production in DCM patients, besides other factors, could be a good target for the use of gene editing.

Immunological-based approaches for cancer therapy.

The immunologic landscape of tumors has been continuously unveiled, providing a new look at the interactions between cancer cells and the immune system. Emerging tumor cells are constantly eliminated by the immune system, but some cells establish a long-term equilibrium phase leading to tumor immunoediting and, eventually, evasion. During this process, tumor cells tend to acquire more mutations. Bearing a high mutation burden leads to a greater number of neoantigens with the potential to initiate an immune response. Although many tumors evoke an immune response, tumor clearance by the immune system does not occur due to a suppressive tumor microenvironment. The mechanisms by which tumors achieve the ability to evade immunologic control vary. Understanding these differences is crucial for the improvement and application of new immune-based therapies. Much effort has been placed in developing in silico algorithms to predict tumor immunogenicity and to characterize the microenvironment via high-throughput sequencing and gene expression techniques. Each sequencing source, transcriptomics, and genomics yields a distinct level of data, helping to elucidate the tumor-based immune responses and guiding the fine-tuning of current and upcoming immune-based therapies. In this review, we explore some of the immunological concepts behind the new immunotherapies and the bioinformatic tools to study the immunological aspects of tumors, focusing on neoantigen determination and microenvironment deconvolution. We further discuss the immune-based therapies already in clinical use, those underway for future clinical application, the next steps in immunotherapy, and how the characterization of the tumor immune contexture can impact therapies aiming to promote or unleash immune-based tumor elimination.

Transcriptional characterization of immunological infiltrates and their relation with glioblastoma patients overall survival.

Introduction: Several cell populations from the peripheral immune system interact to create a complex immunologic status during glioblastoma growth and response to therapy. The aim of this study was to integrate the impact of different immune cell populations present in glioblastoma tumor microenvironment on overall survival. Methodology: Gene expression and clinical data were generated by The Cancer Genome Atlas and previously reported meta-signatures representing cells of the immune system were used. The relationship between meta-signatures was evaluated through Pearson's correlation analyses. Survival analyses were performed through Kaplan-Meier plots and Cox regression model. Results and discussion: Meta-signatures corresponding to infiltrating immune cells with immunosuppressive roles, such as macrophages, NK and NK T cells, MDSCs and Tregs, correlated with poorer patient prognosis. Meta-signatures related to CD8+ T cells predicted improved survival only in patients with low immunosuppressive meta-signatures. By clustering the meta-signatures we found that the cluster containing high meta-signatures of macrophages, MDSCs and Tregs demonstrated the worst prognosis. Conclusion: Integrating the information provided by transcriptional signatures of immunological aspects is fundamental in understanding the impact of the immune system on patient survival. We found a predictive impact on survival with positive role for CD8 and negative roles for macrophages, MDSC, Tregs, NK and NK-T in glioblastoma patients. Understanding these regulatory and stimulatory factors of patients' immune system is essential to delineate an effective strategy to increase the anti-tumor immune response and to generate potential clinical benefits.

Immunological-based approaches for cancer therapy

The immunologic landscape of tumors has been continuously unveiled, providing a new look at the interactions between cancer cells and the immune system. Emerging tumor cells are constantly eliminated by the immune system, but some cells establish a long-term equilibrium phase leading to tumor immunoediting and, eventually, evasion. During this process, tumor cells tend to acquire more mutations. Bearing a high mutation burden leads to a greater number of neoantigens with the potential to initiate an immune response. Although many tumors evoke an immune response, tumor clearance by the immune system does not occur due to a suppressive tumor microenvironment. The mechanisms by which tumors achieve the ability to evade immunologic control vary. Understanding these differences is crucial for the improvement and application of new immune-based therapies. Much effort has been placed in developing in silico algorithms to predict tumor immunogenicity and to characterize the microenvironment via high-throughput sequencing and gene expression techniques. Each sequencing source, transcriptomics, and genomics yields a distinct level of data, helping to elucidate the tumor-based immune responses and guiding the fine-tuning of current and upcoming immune-based therapies. In this review, we explore some of the immunological concepts behind the new immunotherapies and the bioinformatic tools to study the immunological aspects of tumors, focusing on neoantigen determination and microenvironment deconvolution. We further discuss the immune-based therapies already in clinical use, those underway for future clinical application, the next steps in immunotherapy, and how the characterization of the tumor immune contexture can impact therapies aiming to promote or unleash immune-based tumor elimination.

PCA3 long noncoding RNA modulates the expression of key cancer-related genes in LNCaP prostate cancer cells.

Prostate cancer antigen 3 (PCA3) is a prostate-specific long noncoding RNA (lncRNA) involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. To further comprehend the mechanisms by which PCA3 modulates LNCaP cell survival, we characterized the expression patterns of several cancer-related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR cofactors in response to PCA3 silencing. We also aimed to develop a strategy to stably silence PCA3. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) was used to knock down PCA3 in LNCaP cells. The expression of 84 cancer-related genes, as well as those coding for AR cofactors and EMT markers, was analyzed by quantitative real-time PCR (qRT-PCR). LNCaP-PCA3 silenced cells differentially expressed 16 of the 84 cancer genes tested, mainly those involved in gene expression control and cell signaling. PCA3 knockdown also induced the upregulation of several transcripts coding for AR cofactors and modulated the expression of EMT markers. LNCaP cells transduced with lentivirus vectors carrying an shRNA sequence targeting PCA3 stably downregulated PCA3 expression, causing a significant drop (60 %) in the proportion of LNCaP cells expressing the transgene. In conclusion, our data provide evidence that PCA3 silencing modulates the expression of key cancer-related genes, including those coding for AR cofactors and EMT markers. Transducing LNCaP cells with an shRNA sequence targeting PCA3 led to loss of viability of the cells, supporting the proposal of PCA3 knockdown as a putative therapeutic approach to inhibit PCa growth.

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells.

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza's Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.