In Vitro and In Vivo Model for Evaluation of Novel Therapeutic Agents in Patient derived-Lymphoblastic Leukemia (B-ALL)
B-cell Acute Lymphoblastic Leukemia is a hematological malignancy with malignant transformation and proliferation of B-Cell progenitors. Young patients fare well with common therapeutic agents and hematopoietic stem cell transplant, but a subpopulation is chemo-resistant and has high relapse rate. Besides, prognosis in adult patient is worse than younger individuals. Thus, there is an increase need for development of novel and targeted therapeutics. Lack of suitable in vitro culture conditions for primary B-ALL cells severely impairs the evaluation of new therapeutics. We have established a short-term culture system that supports survival of primary B-ALL cells ex vivo for screening of candidate therapeutic agents. Different culture conditions were evaluated, and cell growth/viability was assessed using Cell titer-Glo assay.
Out of 13 primary B-ALL models studied 5 models proliferated and 5 remain stable and viable till day 3. Flow cytometry study showed that all the models stably expressed CD19 and CD20 throughout the assay. The models were tested for sensitivity to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. Heterogeneous dose dependent responses were observed across the models (IC50 17nM - 0.3 µM doxorubicin), (IC50 3 - 79 nM vincristine) indicating a range of relative sensitivity to B-ALL models. Interestingly, BCR-ABL-ve models were resistant to imatinib and BCR-ABL+ve sensitive. Next. to develop patient derived xenograft preclinical B-ALL model, two million PBMC from B-ALL patients (n=14) were intravenously transplanted into sub-lethally irradiated NCG mice. 100% engraftment(n=5,P1) was observed in all the models, but latency of engraftment(>3%) in peripheral blood varied from 15 days to 3.5 months.
The time of passage varied from 40 to 120 days .Flow cytometry analyses showed 30-95% of the bone marrow cells was hCD45+, hCD19+ cells. Splenomegaly was observed and infiltration of 40-98% of B-ALL cells was detected in spleen. Engrafted cells matched the immunophenotype of the corresponding patient. On serial transplantation (n=15) these models showed similar engraftment and immunophenotype. Thus, at Champions we have established a robust in vitro platform for screening new drugs for B-ALL and a passagable patient derived preclinical xenograft model for in vivo drug efficacy study.