Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts

Using a murine bone marrow transplantation model, we evaluated the long-term engraftment of retrovirally transduced bone marrow cells in nonmyeloablated hosts. Male bone marrow was stimulated in a cocktail of interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 hours, then cocultured on the retroviral producer line MDR18.1 for an additional 24 hours. Functional transduction of hematopoietic progenitors was detected in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) colonies. After retroviral transduction, male bone marrow cells were injected into nonablated female mice. Transplant recipients received three TAXOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-month period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, spleen, and peripheral blood showed no evidence of the transduced MDR1 gene. RT-PCR analysis of total bone marrow RNA showed that transcripts from the MDR1 gene were present in a fraction of the engrafted donor cells. These data show functional transfer of the MDR1 gene into nonmyeloablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cells that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo culture of bone marrow induces an engraftment defect concomitantly with progression of cells through S phase, we propose that the cell cycle transit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft

Astrocytic trans-differentiation completes a multicellular paracrine feedback loop required for medulloblastoma tumor growth

The tumor microenvironment (TME) is critical for tumor progression. However, the establishment and function of the TME remain obscure because of its complex cellular composition. Using a mouse genetic system called mosaic analysis with double markers (MADMs), we delineated TME evolution at single-cell resolution in sonic hedgehog (SHH)-activated medulloblastomas that originate from unipotent granule neuron progenitors in the brain. First, we found that astrocytes within the TME(TuAstrocytes) were trans-differentiated from tumor granule neuron precursors (GNPs), which normally never differentiate into astrocytes. Second, we identified that TME-derived IGF1 promotes tumor progression. Third, we uncovered that insulin-like growth factor 1 (IGF1) is produced by tumor-associated microglia in response to interleukin-4 (IL-4) stimulation. Finally, we found that IL-4 is secreted by TuAstrocytes. Collectively, our studies reveal an evolutionary process that produces a multi-lateral network within the TME of medulloblastoma: a fraction of tumor cells trans-differentiate into TuAstrocytes, which, in turn, produce IL-4 that stimulates microglia to produce IGF1 to promote tumor progression