Stroma-Based Versus Stroma-Free Long-Term Survival and Proliferation of Primary Acute Myeloid Leukemia (AML) Cells

Background: Acute myeloid leukemia (AML) is a very aggressive hematological malignancy that carries an overall poor prognosis. High throughput screening of primary (patient-derived) leukemic cells is becoming increasingly valuable as a preclinical tool to help individualize the treatment of relapsed/refractory AML patients. However, the necessity of robust in-vitro culture conditions has often limited the optimization of high throughput drug sensitivity testing. Our lab has previously validated a high throughput assay in 384-well format that utilizes primary leukemic cells maintained in a cocktail of cytokines tested for chemosensitivity and resistance against anti-leukemic agents (Shum et al. Clinical Lymphoma, Myeloma and Leukemia 2013). However, cytokines have not been shown to support long-term proliferation of primary AML cells; nor does it assess drug candidates within the appropriate tumor microenvironment. Stromal cells of the bone marrow microenvironment, however, have been reported to support not only the growth and maintenance of normal hematopoiesis, but also the long-term, in-vitro growth of leukemic cells, while also influencing their response to chemotherapy agents. A comprehensive comparison of primary AML cell growth under our validated cytokine assay and stromal conditions has not previously been undertaken. In this study, we compared the long-term proliferation and phenotypes of primary AML cells in high-throughput format under different stromal conditions against cytokines only. Results from this study would help optimize parameters for a high-throughput drug sensitivity testing comparing the differential response to anti-leukemic agents under cytokine versus stromal conditions.

Methods: The proliferation and immunophenotypes of relapsed/refractory primary AML peripheral blood samples were assessed in 96-well plates under four culture conditions: 1) cell culture medium only 2) in direct contact with HS-5 bone marrow stromal cells 3) in HS-5 conditioned medium or 4) in a cocktail of four cytokines as per our previously validated cytokine-based assay. Cell viability, cell count and immunophenotypes by fluorescently-conjugated antibodies against CD33, CD34, CD38, CD45, CD117, and CD90 (stromal marker) were determined by flow cytometry weekly for 2 weeks.

Results: Our results demonstrated a very heterogeneous response in the growth of primary AML cells among the different patient samples to support with cytokines, HS-5 conditioned medium, and direct contact with HS-5 stromal cells. Overall, primary AML cells continued to expand after 2 weeks in both cytokine and stromal support. The majority of primary AML samples demonstrated higher proliferation with direct contact with stromal cells compared to cytokines only. AML cells growth with cytokines or HS-5 conditioned medium was also comparable. Our initial investigation also revealed one patient sample with a CD34⁺/CD38⁺ predominant phenotype, which demonstrated a > 600-fold expansion within 2 weeks with cytokines only - greater than 3-times the expansion seen by direct contact with HS-5 stromal cells. Immunophenotypes were maintained for all culture conditions at 2 weeks.

Conclusions: Unlike previous studies, which often investigated the long-term proliferation of primary AML cells with both cytokines and stromal support, we showed that cytokine support not only maintained, but can also continue to expand primary AML cells after 2 weeks, independent of stromal support. Consistent with previous studies, we also confirmed that HS-5 stromal cells support long-term in-vitro expansion of primary AML cells, which cannot be substituted by HS-5 conditioned medium. As expected, the majority of primary AML cells showed higher proliferation with direct contact with HS-5 stromal cells compared to cytokine support, but based on one of our patient's sample, which showed a significantly higher expansion with cytokine support only, it is possible that there is a subset of patients whose cell growth respond better to cytokines. This study represents the first direct comparison between the effects of cytokines and two different stromal conditions on the long-term growth of primary AML cells in high throughput format, while providing support that these in-vitro culture conditions can be independently utilized to compare the differential primary AML cell response to anti-leukemic agents.