Analysis of mouse faecal dysbiosis, during the development of cachexia, induced by transplantation with Lewis lung carcinoma cells.

Cachexia (CC) is a complex wasting syndrome that significantly affects life quality and life expectancy among cancer patients. Original studies, in which CC was induced in mouse models through inoculation with BaF and C26 tumour cells, demonstrated that CC development correlates with bacterial gut dysbiosis in these animals. In both cases, a common microbial signature was observed, based on the expansion of Enterobacteriaceae in the gut of CC animals. However, these two types of tumours induce unique microbial profiles, suggesting that different CC induction mechanisms significantly impact the outcome of gut dysbiosis. The present study sought to expand the scope of such analyses by characterizing the CC-associated dysbiosis that develops when mice are inoculated with Lewis lung carcinoma (LLC) cells, which constitutes one of the most widely employed mechanisms for CC induction. Interestingly, Enterobacteriaceae expansion is also observed in LLC-induced CC. However, the dysbiosis identified herein displays a more complex pattern, involving representatives from seven different bacterial phyla, which were consistently identified across successive levels of taxonomic hierarchy. These results are supported by a predictive analysis of gene content, which identified a series of functional/structural changes that potentially occur in the gut bacterial population of these animals, providing a complementary and alternative approach to microbiome analyses based solely on taxonomic classification.

Long-term serial xenotransplantation of juvenile myelomonocytic leukemia recapitulates human disease in Rag2-/-γc-/- mice.

Juvenile myelomonocytic leukemia is a clonal malignant disease affecting young children. Current cure rates, even with allogeneic hematopoietic stem cell transplantation, are no better than 50%-60%. Pre-clinical research on juvenile myelomonocytic leukemia is urgently needed for the identification of novel therapies but is hampered by the unavailability of culture systems. Here we report a xenotransplantation model that allows long-term in vivo propagation of primary juvenile myelomonocytic leukemia cells. Persistent engraftment of leukemic cells was achieved by intrahepatic injection of 1×10(6) cells into newborn Rag2(-/-)γc(-/-) mice or intravenous injection of 5×10(6) cells into 5-week old mice. Key characteristics of juvenile myelomonocytic leukemia were reproduced, including cachexia and clonal expansion of myelomonocytic progenitor cells that infiltrated bone marrow, spleen, liver and, notably, lung. Xenografted leukemia cells led to reduced survival of recipient mice. The stem cell character of juvenile myelomonocytic leukemia was confirmed by successful serial transplantation that resulted in leukemia cell propagation for more than one year. Independence of exogenous cytokines, low donor cell number and slowly progressing leukemia are advantages of the model, which will serve as an important tool to research the pathophysiology of juvenile myelomonocytic leukemia and test novel pharmaceutical strategies such as DNA methyltransferase inhibition.

Comparison of two different laparotomy methods for modeling rabbit VX2 hepatocarcinoma.

AIM: To compare two different laparotomy methods for modeling rabbit VX2 hepatocarcinoma. METHODS: Thirty New Zealand rabbits were randomly divided into two groups: A and B. Group A was assigned a traditional laparotomy method (embedding tumor fragments directly into the liver with tweezers). Group B was subjected to an improved laparotomy method (injection of tumor fragments into the liver through a 15 G syringe needle). The operation time, incision length, incision infection rate, and mortality rate were compared between the two groups after laparotomy. Magnetic resonance imaging (MRI) was performed to evaluate tumor formation rates and the characteristics of the tumors 2 wk after laparotomy. RESULTS: The mean operation times for the two groups (Group A vs Group B) were 23.2 ± 3.4 min vs 17.5 ± 2.9 min (P < 0.05); the incision length was 3.3 ± 0.5 cm vs 2.4 ± 0.6 cm (P < 0.05); and the mortality rate after 2 wk was 26.7% vs 0% (P < 0.05); all of these outcomes were significantly different between the two groups. The incision infection rates in the two groups were 6.7% vs 0% (P > 0.05), which were not significantly different. MRI performed after 2 weeks showed that the tumor formation rates in the two groups were 90.9% vs 93.3% (P > 0.05). These rates were not significantly different between the two groups. The celiac implantation rate and abdominal wall metastasis rate in the two groups were 36.4% vs 13.3% (P < 0.05) and 27.2% vs 6.7% (P < 0.05), respectively, which were significantly different between the two groups. CONCLUSION: The tumor formation rates were not significantly different between the two methods for modeling rabbit VX2 hepatocarcinoma. However, the improved method is recommended because it has certain advantages.

Spontaneous Post-Transplant Disorders in NOD.Cg- Prkdcscid Il2rgtm1Sug/JicTac (NOG) Mice Engrafted with Patient-Derived Metastatic Melanomas.

Patient-derived tumor xenograft (PDTX) approach is nowadays considered a reliable preclinical model to study in vivo cancer biology and therapeutic response. NOD scid and Il2rg-deficient mice represent the "gold standard" host for the generation of PDTXs. Compared to other immunocompromised murine lines, these mice offers several advantages including higher engraftment rate, longer lifespan and improved morphological and molecular preservation of patient-derived neoplasms. Here we describe a spectrum of previously uncharacterized post-transplant disorders affecting 14/116 (12%) NOD.Cg- Prkdcscid Il2rgtm1Sug/JicTac (NOG) mice subcutaneously engrafted with patient-derived metastatic melanomas. Affected mice exhibited extensive scaling/crusting dermatitis (13/14) associated with emaciation (13/14) and poor/unsuccessful tumor engraftment (14/14). In this context, the following pathological conditions have been recognized and characterized in details: (i) immunoinflammatory disorders with features of graft versus host disease (14/14); (ii) reactive lymphoid infiltrates effacing xenografted tumors (8/14); (iii) post-transplant B cell lymphomas associated with Epstein-Barr virus reactivation (2/14). We demonstrate that all these entities are driven by co-transplanted human immune cells populating patient-derived tumor samples. Since the exploding interest in the utilization of NOD scid and Il2rg-deficient mice for the establishment of PDTX platforms, it is of uppermost importance to raise the awareness of the limitations associated with this model. The disorders here described adversely impact tumor engraftment rate and animal lifespan, potentially representing a major confounding factor in the context of efficacy and personalized therapy studies. The occurrence of these conditions in the NOG model reflects the ability of this mouse line to promote efficient engraftment of human immune cells. Co-transplanted human lymphoid cells have indeed the potential to colonize the recipient mouse initiating the post-transplant conditions here reported. On the other hand, the evidence of an immune response of human origin against the xenotransplanted melanoma opens intriguing perspectives for the establishment of suitable preclinical models of anti-melanoma immunotherapy.

The chemokine CCL5 induces CCR1-mediated hyperalgesia in mice inoculated with NCTC 2472 tumoral cells.

Although the expression of the chemokine receptor CCR1 has been demonstrated in several structures related to nociception, supporting the nociceptive role of chemokines able to activate it, the involvement of CCR1 in neoplastic pain has not been previously assessed. We have assayed the effects of a CCR1 antagonist, J113863, in two murine models of neoplastic hyperalgesia based on the intratibial injection of either NCTC 2472 fibrosarcoma cells, able to induce osteolytic bone injury, or B16-F10 melanoma cells, associated to mixed osteolytic/osteoblastic bone pathological features. The systemic administration of J113863 inhibited thermal and mechanical hyperalgesia but not mechanical allodynia in mice inoculated with NCTC 2472 cells. Moreover, in these mice, thermal hyperalgesia was counteracted following the peritumoral (10-30µg) but not spinal (3-5µg) administration of J113863. In contrast, hyperalgesia and allodynia measured in mice inoculated with B16-F10 cells remained unaffected after the administration of J113863. The inoculation of tumoral cells did not modify the levels of CCL3 at tumor or spinal cord. In contrast, although the concentration of CCL5 remained unmodified in mice inoculated with B16-F10 cells, increased levels of this chemokine were measured in tumor-bearing limbs, but not the spinal cord, of mice inoculated with NCTC 2472 cells. Increased levels of CCL5 were also found following the incubation of NCTC 2472, but not B16-F10, cells in the corresponding culture medium. The intraplantar injection of CCL5 (0.5ng) to naïve mice evoked thermal hyperalgesia prevented by the coadministration of J113863 or the CCR5 antagonist, d-Ala-peptide T-amide (DAPTA), demonstrating that CCL5 can induce thermal hyperalgesia in mice through the activation of CCR1 or CCR5. However, contrasting with the inhibitory effect evoked by J113863, the systemic administration of DAPTA did not prevent tumoral hyperalgesia. Finally, the peritumoral administration of an anti-CCL5 antibody completely inhibited thermal hyperalgesia evoked by the inoculation of NCTC 2472 cells.

Donor cell leukemia in umbilical cord blood transplant patients: a case study and literature review highlighting the importance of molecular engraftment analysis.

Donor cell neoplasms are rare complications of treatment regimens that involve stem cell transplantation for hematological malignancies, myelodysplastic processes, or certain genetic or metabolic disorders. We report a case of donor cell leukemia in a pediatric patient with a history of acute myeloid leukemia that manifested as recurrent AML FAB type M5 fourteen months after umbilical cord blood transplantation. Although there was some immunophenotypic drift from the patient's original AML and their posttransplant presentation, the initial pathological impression was of recurrent disease. Bone marrow engraftment analysis by multiplex PCR of short tandem repeat markers performed on the patient's diagnostic specimen showed complete engraftment by donor cells, with a loss of heterozygosity in the donor alleles on chromosome 7. This led to the reinterpretation of this patient's disease as donor-derived leukemia. This interpretation was supported by a routine karyotype and fluorescence in situ hybridization analysis showing loss of chromosome 7 and a male (donor) chromosome complement in this female patient. Also noted was a loss of the patient's presenting chromosomal abnormality, t(11;19)(q23;p13). This case highlights the need for close coordination between all aspects of clinical testing for the transplant patient, including molecular engraftment studies, when distinguishing the very common complication of recurrent disease from the exceedingly rare complication of donor cell leukemia.

A novel model measuring the harm of transplanting hepatocellular carcinoma exceeding Milan criteria.

No empirical studies have defined the posttransplant survival that would justify expansion of the Milan criteria for liver transplantation of hepatocellular carcinoma. We created a Markov model comparing the survival benefit of transplantation for a patient with >Milan HCC, versus the harm caused to other patients on the waiting list. In the base-case analysis, the strategy of transplanting the patient with >Milan HCC resulted in a 44% increased risk of death and a utility loss of 3 quality-adjusted years of life across the pre- and posttransplant periods for a nationally representative cohort of patients on the waiting list. This harm outweighed the benefit of transplantation for a patient with >Milan HCC having a 5-year posttransplant survival of less than 61%. This survival threshold was most sensitive to geographic variations in organ shortage, with the threshold varying from 25% (Region 3) to >72% (Regions 1, 5, 7 and 9). In conclusion, expansion of the Milan criteria will require demonstrating high survival rates for the newly eligible patients-approximately 61% at 5 years after transplantation. In regions with less severe organ shortage, a more aggressive approach to transplanting these patients may be justified.

Needle tract implantation of follicular neoplasm after fine-needle aspiration biopsy: report of a case.

We herein report a 28-year-old woman with a follicular neoplasm showing subcutaneous needle tract implantation. One month after fine-needle aspiration biopsy (FNAB) for a tumor measuring 2.5 cm, the patient became aware of a subcutaneous nodule measuring about 1 cm at the needle insertion site. FNAB smear of this nodule showed poorly cohesive clusters of follicular cells with nuclear crowding, overlapping and resetting with some microfollicular architecture. Total thyroidectomy and resection of the subcutaneous nodule were performed. Although there was no capsular or vascular invasion of the nodule, the lesion was diagnosed as follicular carcinoma because of the subcutaneous seeding. Ki-67 labeling indices of the thyroid nodule and implanted tumor were higher than 5%. Furthermore, although galectin-3 was completely negative in the thyroid nodule, it was heterogeneously positive in the implanted tumor. It is therefore suggested that high cell proliferating activity as a characteristic of the original nodule and the subsequently obtained invasive characteristic of the implanted tumor contributed to this event. To date, there has not been any recurrence of the implanted lesion. Because implanted follicular carcinoma can be surgically removed, this complication should not impair the usefulness of FNAB.

The Tel-Abl (ETV6-Abl) tyrosine kinase, product of complex (9;12) translocations in human leukemia, induces distinct myeloproliferative disease in mice.

Several patients with clinical features of chronic myeloid leukemia (CML) have fusion of the TEL (ETV6) gene on 12p13 with ABL on 9q34 and express a chimeric Tel-Abl protein that contains the same portion of the Abl tyrosine kinase fused to Tel, an Ets family transcription factor, rather than Bcr. In a murine retroviral bone marrow transduction-transplantation model, a Tel (exon 1-5)-Abl fusion protein induced 2 distinct illnesses: a CML-like myeloproliferative disease very similar to that induced by Bcr-Abl but with increased latency and a novel syndrome characterized by small-bowel myeloid cell infiltration and necrosis, increased circulating endotoxin and tumor necrosis factor alpha levels, and fulminant hepatic and renal failure. Induction of both diseases required the Tel pointed homology oligomerization domain and Abl tyrosine kinase activity. Myeloid cells from mice with both diseases expressed Tel-Abl protein. CML-like disease induced by Tel-Abl and Bcr-Abl was polyclonal and originated from cells with multilineage (myeloid, erythroid, and B- and T-lymphoid) repopulating ability and the capacity to generate day-12 spleen colonies in secondary transplantations. In contrast to findings with Bcr-Abl, however, neither Tel-Abl-induced disease could be adoptively transferred to irradiated secondary recipient syngeneic mice. These results show that Tel-Abl has leukemogenic properties from distinct from those of Bcr-Abl and may act in a different bone marrow progenitor.

Development of port-site metastasis after pneumoperitoneum.

BACKGROUND: Port-site metastasis is a critical problem in laparoscopic cancer surgery; the pathogenesis and means of prevention are still unclear. The aim of this study was to clarify by scanning electron microscopy the initial morphologic changes in the development of port-site metastasis. METHODS: Fifteen nude mice were injected with human gastric cancer (MKN 45) cells. Mice were killed on days 0, 3, and 8 (n = 5 each day) after intraperitoneal injection of 5 x 105 cancer cells and carbon dioxide (CO2) pneumoperitoneum at 4-6 mmHg for 20 min. The abdominal wall with the port sites was harvested and examined under both light and scanning electron microscopy. RESULTS: Immediately after CO2 pneumoperitoneum (day 0), the abdominal peritoneum was peeled away and the muscular layer was destroyed at the port site in all mice. Several cancer cells were attached to the injured port sites. On day 3, the subperitoneal tissue and muscular layer defects were replaced by granulation tissue, and several cancer cells were observed in the subperitoneal tissue. On day 8, a small nodule was macroscopically visible at the port site; it was completely covered by mesothelial cells and consisted of numerous cancer cells. CONCLUSIONS: Free cancer cells appear to attach to the injured port sites immediately after CO2 pneumoperitoneum, and these are associated with the development of port-site metastasis after laparoscopic cancer surgery.

Frequent development of murine T-cell lymphomas with TcR alpha/beta+, CD4-/8- phenotype after implantation of human inflammatory breast cancer cells in BALB/c nude mice.

Tumors developed quite frequently in some of the visceral organs, including spleen and liver, in BALB/c nude mice upon subcutaneously xenografting surgical specimens from five different inflammatory breast cancer patients. All of these tumors developed within two and a half months to one year after the subcutaneous inoculation of surgical specimens. From these tumors, five independent transplantable tumors, including tMK-2, tHK-1, tYK-1, tYK-2 and tTY-1 have been established. Chromosome analysis, morphologic studies by light and electron microscopy and phenotype analysis indicated that these tumors are of mouse origin. The tMK-2 tumor was highly metastatic to the spleen and liver when it was subcutaneously transplanted into the right scapular region. In addition, the region where the tMK-2 tumor cells were subcutaneously inoculated showed an apparently inflammatory process represented by erythema. After subcutaneous inoculation into the right scapular region, tHK-1, tYK-1, 2, and tTY-1 tumors also metastasized to some of the visceral organs, including spleen and liver. From these tumors, in vitro cell lines were established. The cells grew in a stromal-cell dependent manner under in vitro culture conditions. The cells were again tumorigenic at the inoculated region and metastasized to various organs, including liver and spleen, of BABL/c nude mice. Histological examination revealed that the tumors showed features of malignant lymphoma. Phenotypically, these five tumors expressed early T lymphocyte markers as revealed by anti-mouse anti-TcR alpha/beta, anti-CD3, CD4 and CD8 monoclonal antibodies. To our knowledge, these cell lines are the first T-cell lines showing the phenotype of extrathymically differentiated T-cells in the liver.

[Gradually diminishing tumor protection caused by reimplants]. / Bradifilaxia tumoral por reimplantes.

Mice from which a MCA-induced sarcoma has been removed and which are exposed to repeated (every three months) tumoral cell transplants, gradually lose their protection against a certain threshold number of cells. Although the survival period after each transplant is longer than in non-protected animals (those that never received a primary tumor) it is seen that while some of them survive for three months (these are the ones to be re-inoculated with tumoral cells) others die. The proportion of mice which die rises with the number of inoculations received; and among those which die, the proportion of mice without localized tumor or neoplastic dissemination is also progressively higher. We do not know why these mice die at a later and cachectic stage without tumor but in a situation resembling a GVH (graft versus host) reaction. Repeated challenge through re-inoculation induces "bradyphylaxis" (progressively diminishing protection). On histopathological examination intense congestion is found, with haemorrhages in the lungs, liver, spleen and kidneys.