An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host ...sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p = 2.5 × 10(-6)), and we observed a positive association with lymph node metastasis.
High‐grade serous carcinoma (HGSC) is the most common and fatal form of ovarian cancer. While most tumours are highly sensitive to cytoreductive surgery and platinum‐ and taxane‐based chemotherapy, ...the majority of patients experience recurrence of treatment‐resistant tumours. The clonal origin and mutational adaptations associated with recurrent disease are poorly understood. We performed whole exome sequencing on tumour cells harvested from ascites at three time points (primary, first recurrence, and second recurrence) for three HGSC patients receiving standard treatment. Somatic point mutations and small insertions and deletions were identified by comparison to constitutional DNA. The clonal structure and evolution of tumours were inferred from patterns of mutant allele frequencies. TP53 mutations were predominant in all patients at all time points, consistent with the known founder role of this gene. Tumours from all three patients also harboured mutations associated with cell cycle checkpoint function and Golgi vesicle trafficking. There was convergence of germline and somatic variants within the DNA repair, ECM, cell cycle control, and Golgi vesicle pathways. The vast majority of somatic variants found in recurrent tumours were present in primary tumours. Our findings highlight both known and novel pathways that are commonly mutated in HGSC. Moreover, they provide the first evidence at single nucleotide resolution that recurrent HGSC arises from multiple clones present in the primary tumour with negligible accumulation of new mutations during standard treatment.
The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related ...tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.
The plasticity of pancreatic tissue is demonstrated in many pancreatic diseases. It has previously been shown that pancreatic islet-to-duct transformation and acinoductal metaplasia have been ...associated with both pancreatic regeneration and adenocarcinoma in various in vivo and in vitro settings. Understanding this inherent morphogenetic plasticity of the adult pancreas could lead to new therapeutic approaches to pancreatic disease.
Cadaveric human pancreases (n = 7) were digested, and purified acinar tissue, which was approximately 85% immunoreactive for amylase and approximately 15% immunoreactive for CK-19, was embedded in a type 1 collagen matrix and cultured in a differentiation medium (DM) consisting of Dulbecco modified Eagle/F12 medium supplemented with cholera toxin (100 ng/mL), epidermal growth factor (10 ng/mL), and insulin (24 mU/mL) for 8 days. After this initial period, the resulting tissues were cultured in DM without cholera toxin, supplemented with gastrin (50 nmol/L) and hepatocyte growth factor (HGF; 10 ng/mL), with islet neogenesis-associated protein (INGAP; 167 nmol/L) or with gastrin + HGF + INGAP for 6 days. Tissue samples were then analyzed for amylase, cytokeratin 19, pancreas duodenum homeobox 1, and endocrine hormone immunoreactivity as well as dithizione positivity.
After 8 days of culture, approximately 90% of acini transformed into ductlike structures. This acinoductal transformation was characterized by a complete absence of amylase staining, with virtually all cells CK-19 immunoreactive. Addition of INGAP led to an approximately 18-fold increase in pancreas duodenum homeobox 1 immunoreactivity, although without an observed increase in insulin production as measured by dithizone positivity. However, when acinar-derived ductlike structures were cultured with gastrin + HGF + INGAP, the total incidence of dithizone-positive structures increased approximately 6-fold (10.9 +/- 2.9% vs 1.7 +/- 0.4%, P = 0.037). Treatment with gastrin + HGF alone led to no significant change in any of the measured parameters.
We have developed a novel in vitro model of adult human acinoductal metaplasia that will aid not only in developing new methods of expanding beta-cell mass but also provide insights into pancreatic carcinogenesis.
T-cell antigen receptor (TCR) variability enables the cellular immune system to discriminate between self and non-self. High-throughput TCR sequencing (TCR-seq) involves the use of next generation ...sequencing platforms to generate large numbers of short DNA sequences covering key regions of the TCR coding sequence, which enables quantification of T-cell diversity at unprecedented resolution. TCR-seq studies have provided new insights into the healthy human T-cell repertoire, such as revised estimates of repertoire size and the understanding that TCR specificities are shared among individuals more frequently than previously anticipated. In the context of disease, TCR-seq has been instrumental in characterizing the recovery of the immune repertoire after hematopoietic stem cell transplantation, and the method has been used to develop biomarkers and diagnostics for various infectious and neoplastic diseases. However, T-cell repertoire sequencing is still in its infancy. It is expected that maturation of the field will involve the introduction of improved, standardized tools for data handling, deposition and statistical analysis, as well as the emergence of new and equivalently large-scale technologies for T-cell functional analysis and antigen discovery. In this review, we introduce this nascent field and TCR-seq methodology, we discuss recent insights into healthy and diseased TCR repertoires, and we examine the applications and challenges for TCR-seq in the clinic.
Off-tumor targeting of human antigens is difficult to predict in preclinical animal studies and can lead to serious adverse effects in patients. To address this, we developed a mouse model with ...stable and tunable human Her2 (hHer2) expression on normal hepatic tissue and compared toxicity between affinity-tuned Her2 chimeric antigen receptor T cells (CARTs). In mice with hHer2-high livers, both the high-affinity (HA) and low-affinity (LA) CARTs caused lethal liver damage due to immunotoxicity. In mice with hHer2-low livers, LA-CARTs exhibited less liver damage and lower systemic levels of IFN-γ than HA-CARTs. We then compared affinity-tuned CARTs for their ability to control a hHer2-positive tumor xenograft in our model. Surprisingly, the LA-CARTs outperformed the HA-CARTs with superior antitumor efficacy in vivo. We hypothesized that this was due, in part, to T cell trafficking differences between LA and HA-CARTs and found that the LA-CARTs migrated out of the liver and infiltrated the tumor sooner than the HA-CARTs. These findings highlight the importance of T cell targeting in reducing toxicity of normal tissue and also in preventing off-tumor sequestration of CARTs, which reduces their therapeutic potency. Our model may be useful to evaluate various CARTs that have conditional expression of more than 1 single-chain variable fragment (scFv).
Chimeric antigen receptor (CAR) engineered T cells have been used clinically to improve outcomes in patients with hematopoietic malignancies owing to the ability of CAR T cells to recognize tumor ...antigens and kill malignant cells. CAR T cells possess the antigen recognizing capability of an antibody through the single chain variable fragment (scFv) and their cytotoxicity is enhanced through signaling via the intracellular domains of T cell receptors and co-activating receptors such as CD3zeta and 4-1BB, respectively. Thus, CAR expressing T cells are able to detect cancer cells through tumor antigens and can become activated to unleash their cytotoxic potentials in a non-MHC restricted manner. Therapeutic side-effects can occur when T-cell receptor targeting is misdirected to the incorrect tissue causing potentially serious on-target off-tumor cytotoxicity. Factors that influence CAR targeting include expression levels of tumor-associated antigen in normal tissue and the binding affinities of scFvs.
Our first step in developing an in vivo, on target, off-tumor, CAR T cell toxicity model was to generate mice with tunable expression of a human tumor antigen in normal tissue. NSG mice were IV injected with recombinant adeno-associated virus serotype 8 (rAAV8) to deliver a truncated human ErbB2 (Her2/neu or CD340) gene and a Katushka fluorescent reporter that were driven by the liver-specific promoter, thyroxine binding globulin (TBG). AAV8 genomic copies (GCs) were injected at varying dilutions of 1.5 x 1012 GC/mouse, 7.25 x 1011 GC/mouseand 1.5x1010 GC/mouse to induce a range of expression of ErbB2 in the liver. Katushka expression was visualized in vivo using the IVIS small animal imager. ErbB2 gene expression was detected using reverse transcription polymerase chain reaction (qRT-PCR) and the ErbB2 protein was detected using western blots and immunohistochemistry (IHC). Our data has shown that expression levels of ErbB2 and the Katushka reporter positively correlated with the number of AAV8 GCs that were injected. This enabled us to obtain ErbB2 expression levels in the liver comparable to the levels seen in either ErbB2High tumors (eg. SK-OV3) or ErbB2Low tumors (eg. PC3 and HEK293T).
To determine if affinity tuning of scFvs will allow CAR T cells to discriminate between high and low ErbB2 expression in the liver, T cells were engineered to co-express the click beetle red (CBR) reporter and either a high-affinity scFv, anti-ErbB2 CAR (4D5) or a low-affinity scFv, anti-ErbB2 CAR (4D5-5). These T cells were then IV injected into NSG mice that had either high or low ErbB2-expressing livers. Although these experiments were ongoing at the time of abstract submission, we will show our results on T cell trafficking in the liver, which will be visualized by IHC and by in vivo imaging using the IVIS small animal imager. Liver toxicity will be assessed by histological examination and by measuring liver function via standard enzymatic testing of blood.
Furthermore, we aim to show whether affinity tuning of scFvs will allow CAR T cells to selectively recognize and target ErbB2High tumors while sparing ErbB2Low normal tissue. This will be performed by inoculating ErbB2high SK-OV3 tumor cells into mice with ErbB2Low livers followed by IV injection with either 4D5 or 4D5-5 CAR T cells. We expect that the low-affinity anti-ErbB2 CAR (4D5-5) T cells will target the ErbB2High SK-OV3 tumor cells and cause tumor regression while preserving function in the ErbB2Low liver. If so, then we will have shown that our pre-clinical mouse model can be used to identify on-target off-tumor CAR T cell toxicity, which will aid in improving the safety profile and clinical outcomes of future CAR T cell therapies.
Scholler:Novartis: Patents & Royalties. Zhao:Novartis: Patents & Royalties, Research Funding. June:Novartis: Patents & Royalties, Research Funding.
The regenerating (
Reg) genes are associated with tissue repair and have been directly implicated in pancreatic β-cell regeneration.
A hamster Reg3, Islet neogenesis associated protein (INGAP), has ...been shown to possess anti-diabetic properties in rodent models. Although several
Reg3 proteins have been identified in other species, INGAP is the only
Reg3 found in hamsters. To identify new
Reg3 genes in the hamster pancreas we employed homology reverse transcription polymerase chain reaction (RT-PCR) using degenerate
Reg3 primers, followed by rapid amplification of cDNA ends (RACE). We report here the discovery of a new hamster
Reg3 gene of 765 nucleotides (nt) that encodes a 174-amino acid (aa) protein. This protein sequence was identified as a novel hamster
Reg3γ with 78% and 75% identity to the rat
Reg3γ and mouse
Reg3γ protein, respectively. We also fully sequenced the previously reported partial sequence of the hamster
Reg1 gene coding region using RACE to yield a 756-nt transcript that encodes a deduced 173 aa protein. This protein was identified as hamster
Reg2, rather than
Reg1 as was initially reported, with an 81% identity to mouse
Reg2. The spatial gene expression patterns of the hamster Reg genes, analyzed by RT-PCR, were similarly distributed with low level expression being found globally throughout the body. Mice and hamsters are the only species known to carry either of the functional
INGAP or
Reg2 genes. It remains to be determined whether these genes bestow mice and hamsters with special regenerative abilities in the pancreas.
Recent successes in islet transplantation highlight the importance of islet isolation by experienced centers and minimization of cell injury as crucial to the achievement of insulin independence. ...Islet injury may manifest as cell death by apoptosis, shorter graft survival, and the need for retransplantation. Although an inflammatory cytokine response at the graft site is known to inhibit engraftment, recent evidence indicates that islet cells may contribute to this response.
Isolated human islets were cultured for up to one week in serum-free CMRL-1066 with 25 microM of tumor necrosis factor (TNF)alpha inhibitor RDP58. Gene expression was measured by reverse transcriptase polymerase chain reaction, apoptosis and TNFalpha secretion by enzyme-linked immunosorbent assay and enzyme-linked immunospot, and islet function by stimulated insulin secretion.
Isolation induced a twofold increase in TNFalpha expression between days one and three (P<0.05), while TNFalpha secretion peaked at day one. RDP58 reduced TNFalpha secretion by 70.6% (P<0.02), though TNFalpha gene expression was unaffected. RDP58 reduced the frequency of TNFalpha-secreting islets by 64.4% (P<0.05) and reduced apoptotic levels by 26.4% within 24 hr postisolation (P<0.05). The reduction in apoptosis was maintained throughout the week (P<0.01), while apoptosis increased in control cultures. Finally, RDP58-treated islets displayed increased insulin secretion in response to both elevated glucose (1915.0+/-396.6 vs. 825.3+/-261.1 mU/L, P<0.01) and secretagogues (2294.3+/-529.5 vs. 939.8+/-333.7 mU/L, P<0.02).
These data demonstrate that intraislet cytokine production should be considered as a factor leading to islet cell death postisolation and postengraftment, and strategies aimed at countering islet cytokine production represent a novel target for improving islet viability and function.
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