Chronic lymphocytic leukemia (CLL) cells require complex microenvironmental and immunologic interactions to survive and proliferate. Such interactions might be best recreated in animal models; however, this needs extensive verification. We therefore investigated the composition of the T-cell compartment in the El-TCL1 transgenic mouse, currently the most widely used murine model for CLL. Immunophenotyping and transplant approaches were used to define T-cell subsets at various stages of CLL. Analogous to human CLL, we observed a skewing of T-cell subsets from naive to antigen-experienced memory T cells that was more pronounced in lymph nodes than in blood. Transplantation of CLL into non-transgenic recipients was feasible without immunosuppression in a pure C57BL/6 background and resulted in the prominent skewing of the T cells of the recipient mice. Both in spontaneously developed CLL and in the transplantation setting, a loss in T-cell receptor diversity was observed, with a relevant number of clonal T-cell populations arising. This suggests that antigen-dependent differentiation toward the T memory pool is initiated by murine CLL cells. In summary, we validate the TCL1 transgenic mouse model for analysis of T-cell phenotypes and suggest a CLL-dependent antigen-driven skewing of T cells in these mice.

IntroductionThe B-cell receptor (BCR) is proposed to generate vital signals in the pathogenesis of chronic lymphocytic leukemia (CLL). Specifically in cases with unmutated BCR sequences, crosslinking of the BCR seems to contribute to the survival and proliferation of the CLL cells. 1 The observed bias in V-gene usage and the nonrandom nature of rearranged CDR3 sequences in CLL also suggest that BCR signaling is involved in the initial selection and development of the malignant clone. 2 Reports have linked the activity of members of the protein kinase C family with the pathogenesis of CLL. [3][4][5][6][7] Protein kinase C (PKC) consists of at least 10 isoenzymes with different activation requirements and substrate specificities. Several of these have been associated with diverse cancer settings. 8 PKC␣ and PKC␦ have been indirectly associated with CLL. 5,7 PKC␤ is involved in the transduction and fine-tuning of BCR signals in normal B cells, 9 and it has recently been implicated in CLL pathogenesis. Studies have shown an overexpression of PKC␤ in human CLL and link the signaling downstream of PKC␤ with other relevant signaling events, such as calcium flux, or PKB/AKT phosphorylation. 3,4 However, to define the role of PKC␤, these studies used inhibitors that are at best only relatively specific for the enzyme, thus warranting genetic evidence for its contribution to CLL pathogenesis. Although PKC␤ is shown to be part of a poor-prognosis gene cluster in CLL linked to the transmission of BCR signals, 10 its role in regulating cell survival is more complex. Depending on overall PKC␤ expression, either enhanced or decreased survival was observed, 3 suggesting that PKC␤ signal strength may determine the outcome. Interestingly, targeted deletion of PKC␤ in a murine model led to a preferential loss of CD5 ϩ B cells, 9 suggesting that in these cells PKC␤ provides an essential developmental signal.We used the TCL1 transgenic mouse model for human unmutated CLL to determine the role of PKC␤ in a clean genetic targeting experiment. 11,12 CLL cells in these mice display unmutated BCR sequences with a bias regarding the V-gene usage, suggesting a role of antigen-dependent selection also in the mouse model. Importantly, the premalignant stage of CD5 ϩ B-cell hyperplasia lends itself to the exploration of initial stages of the disease. 11 We crossed TCL1 transgenic mice with PKC␤ knockout mice to assess the effect of PKC␤ deletion on the development of both the premalignant phenotype and CLL onset. In addition, we used a novel PKC␤ inhibitor, enzastaurin, currently in clinical development, to show the potential of translating our murine findings into the human system (for a review on the substance see Ma and Rosen 13 ). MethodsMice E-TCL1 transgenic mice 11 and PKC␤ knockout mice 9 were provided by C. Croce (Columbus, Ohio) and M. Leitges (Oslo, Norway), respectively. Both strains have been backcrossed for 9 generations to C57BL/6 mice. Genotyping of mice was performed on tail DNA by polymerase chain reaction (PCR) as previousl...

To date, cancer is still the second most prevalent cause of death after cardiovascular diseases in the industrialized word, whereby the primary cause of cancer is not attributed to primary tumor formation, but rather to the growth of metastases at distant organ sites. For several years it was considered that the well-known phenomenon of organ-specific spreading of tumor cells is mostly a mechanical process either directed passively due to size constraints (mechanical trapping theory) or due to a fertile environment provided by the organ in which tumor cells can proliferate (seed and soil hypothesis). Both mechanisms strongly depend on the adhesive properties of tumor cells either to endothelial cells and/or cancer cells, which are facilitated by a variety of cell adhesion molecules including carbohydrates and integrins. Within the past years it became evident that the organ-specific metastatic spreading of tumor cells does not only rely on heterotypic and homotypic adhesive interactions, but also on the interplay of chemokines and their appropriate receptors. Moreover, the identification of cancer stem cells in various tumor tissues has opened new questions. Cancer stem cells possess self-renewal, differentiation, and tumor-initiating capacities. Thus these cells are ideal candidates to be the seed of a secondary tumor. In the present review we will give a brief overview about the complex process of organ-specific metastasis formation depending on the interplay of adhesion molecules, chemokines, and the putative role of cancer stem cells in metastasis formation.

The abilities of tumor cells to extravasate from the blood vessel system and to migrate through the connective tissue are prerequisites in metastasis formation. Both processes are chiefly mediated by integrins, which mediate both cell-cell and cell-matrix interactions. We investigated the role of integrin subunits in the adhesion, extravasation and migration of the highly invasive human bladder carcinoma cell line T24. Here we show that inhibition of the beta(1)-integrin subunit using the specific beta(1)-integrin blocking antibody 4B4 significantly reduces the adhesion to HUVEC and transmigratory activity of T24 cells. The blockade of the beta(1)-integrin subunit also resulted in a significantly reduced locomotory activity of T24 cells. A detailed cell migration analysis on a single cell level revealed that blockade of the beta(1)-integrin subunit leads to an altered migration pattern of single cells but does not influence migration per se. Migration parameters such as time active, velocity and distance migrated were significantly reduced as compared to untreated control cells. Our observations strongly suggest a central role for the beta(1)-integrin subunit in forming the cell-cell and cell-matrix bonds necessary for adhesion, extravasation and migration.

Realtime laser scanning confocal microscopy leads to a better understanding of the complex and dynamic cell-to-cell and cell-to-matrix interactions during the extravasation process.