Humanized Mouse Model of Ovarian Cancer Recapitulates Patient Solid Tumor Progression, Ascites Formation, and Metastasis

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγnull (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma

Safety of procuring research tissue during a clinically indicated kidney biopsy from patients with lupus: data from the Accelerating Medicines Partnership RA/SLE Network

Objectives In lupus nephritis the pathological diagnosis from tissue retrieved during kidney biopsy drives treatment and management. Despite recent approval of new drugs, complete remission rates remain well under aspirational levels, necessitating identification of new therapeutic targets by greater dissection of the pathways to tissue inflammation and injury. This study assessed the safety of kidney biopsies in patients with SLE enrolled in the Accelerating Medicines Partnership, a consortium formed to molecularly deconstruct nephritis.Methods 475 patients with SLE across 15 clinical sites in the USA consented to obtain tissue for research purposes during a clinically indicated kidney biopsy. Adverse events (AEs) were documented for 30 days following the procedure and were determined to be related or unrelated by all site investigators. Serious AEs were defined according to the National Institutes of Health reporting guidelines.Results 34 patients (7.2%) experienced a procedure-related AE: 30 with haematoma, 2 with jets, 1 with pain and 1 with an arteriovenous fistula. Eighteen (3.8%) experienced a serious AE requiring hospitalisation; four patients (0.8%) required a blood transfusion related to the kidney biopsy. At one site where the number of cores retrieved during the biopsy was recorded, the mean was 3.4 for those who experienced a related AE (n=9) and 3.07 for those who did not experience any AE (n=140). All related AEs resolved.Conclusions Procurement of research tissue should be considered feasible, accompanied by a complication risk likely no greater than that incurred for standard clinical purposes. In the quest for targeted treatments personalised based on molecular findings, enhanced diagnostics beyond histology will likely be required

Transitional B cells in quiescent SLE: An early checkpoint imprinted by IFN

Systemic lupus (SLE) is characterized by a break of B cell tolerance that plays a central role in disease pathophysiology. An early checkpoint defect occurs at the transitional stage leading to the survival of autoreactive B cells and consequently the production of pathogenic autoantibodies. The main purpose of our work was to determine whether transitional B cells, as the most immature naïve B cell subset upstream of pathogenic B cells, display specific features compared to healthy non SLE subjects. Through extensive analysis of transitional B cells from untreated or low treated, mostly Caucasian, SLE patients, we demonstrated that transitional (T1 and T2) B cell frequencies were increased in SLE and positively correlated with disease activity. SLE transitional B cells displayed defects in two closely inter-related molecules (i.e. TLR9 defective responses and CD19 downregulation). RNA sequencing of sorted transitional B cells from untreated patients revealed a predominant overexpression of interferon stimulated genes (ISGs) even out of flares. In addition, early transitional B cells from the bone marrow displayed the highest interferon score, reflecting a B cell interferon burden of central origin. Hence, the IFN signature in transitional B cells is not confined to African American SLE patients and exists in quiescent disease since the medullary stage. These results suggest that in SLE these 3 factors (i.e. IFN imprintment, CD19 downregulation and TLR9 responses impairment) could take part at the early transitional B cell stage in B cell tolerance by-pass, ultimately leading in periphery to the expansion of autoantibodies-secreting cells

Evidence of Viability and Function of T Cells and Plasma Cells in Tumor Bearing Mice.

a<p>NSG mice injected i.p. with control empty liposomes or liposomes loaded with IL-12. The dose of IL-12 was 50 µg/mouse. All mice were treated 21 days after i.p. injection of the tumor-derived cell aggregates.</p>b<p>Mice were bled 5 days after treatment, and the serum levels of IFN-γ determined by ELISA. Each value represents the serum level from a single mouse.</p>c<p>Mice were bled 78 days after treatment and the serum levels of human immunoglobulin determined by ELISA. Each value represents the serum level from a single mouse.</p

Presence of tumor, T cells and fibroblasts in tumor cell aggregates.

<p>Histology and immunohistochemistry of tumor-derived cell aggregates derived by mild disruption of a primary ovarian tumor. H&E staining show clusters of cells of different sizes (A) Immunohistochemical staining for human CD45 (B) and CD3 (C) shows evidence of human leukocytes and T cells, i.e. dark brown stained cells. Trichrome staining (D) reveals the presence of collagen which is produced by fibroblasts and stains aquamarine. Tumor cells stain dark brown with immunohistochemical stain for cytokeratin (E). All figures are at 400× magnification.</p

Frequency of Tumor Development Following i.p. Injection of Tumor Cell Aggregates Derived from Ovarian Cancer Patient Solid Tumors.

a<p>Each experiment was conducted with tumor tissue derived from a different ovarian cancer patient. In experiment 5A and B the cell aggregates was generated from the same tumor.</p>b<p>Mice were injected i.p. with tumor-derived cell aggregates obtained from five different ovarian cancer patients, that is generated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024420#s4" target="_blank">Materials and Methods</a> section.</p>c<p>Evidence of tumor was established by the presence of grossly detected tumor nodules in the peritoneal cavity, histological evidence of tumor in the organs or both. In each experiment, #1–4, one of the 10 mice died without evaluation. In experiment #5 one of the mice was sacrificed at day 85 post inoculation without any evidence of gross tumor. In experiment 5B – one of the mice died without evaluation.</p

Spontaneous metastasis of tumor xenograft into the lung.

<p>H&E staining (100× magnification) reveals the presence of tumor in the diaphragm (A), and in the lung 100× magnification (B) and 400× magnification (C). The immunohistochemical staining of the tumor in the lung for HLA Class I (D) establishes that these tumors are of human origin. The sections were made from the tissues of a mouse 16 weeks after the i.p. injection of a suspension of tumor = derived cell aggregates.</p

Tumor and stroma in multiple organs in NSG mice following injection of tumor aggregates.

<p>H&E staining shows evidence of tumor (see arrows) in peritoneal nodule from omentum (Aa and Ab), ovary and periovarian fat (Ac), pancreas (Ad), uterus (Ae), spleen (Af), and liver (Ag). These tumors resulted from the i.p. injection of tumor-derived cell aggregates derived from a papillary serous carcinoma of the ovary. Mice were sacrificed 140 days post inoculation. Lymphocytes (arrow) were observed in juxtaposition with tumor cell (arrowhead) (Ba). Immunohistochemical staining (B) shows the presence of human CD45+ leukocytes (Bb), CD3+ T cells (Bc), CD20+ B cells (Bd), CD138+ plasma cells (Be), and HLA+ tumor nodule adjacent to the ovary (Bf). Proliferation of tumor cells is shown by positive stain with KI67 (Bg), and evidence of stromal fibroblasts illustrated by trichrome staining of collagen see arrows (Bh). Arrow head shows tumor cells. All sections in A are at 100× magnification and in B the sections are at 400× magnification.</p

Histology of the original tumor.

<p>A section of a papillary serous adenocarcinoma of the ovary stained with hemotoxylin and eosin (H&E). Shown in A 100× magnification and in B 400× magnification clusters of tumor cells (T) that show lymphocytic infiltration (L), along with fibrous connective tissue (F).</p